Several studies indicate that at the choriodecidual interface, where maternal and fetal tissues make contact, a network of signals is established during labor that includes infiltration of leukocytes and secretion of pro-inflammatory cytokines. In this review, we provide an overview of the inflammatory milieu present in the choriodecidua during membrane rupture, describe the recruitment and homing of leukocytes to the reproductive tissues, and detail specific actions of the key chemokines released by the choriodecidual cells. These data lend further support to the hypothesis that labor is an inflammatory response, wherein the infiltrated leukocytes in the choriodecidua interface could be contributing to the creation of a microenvironment leading to collagenolysis, which would promote the rupture of these tissues during labor. In addition to the available information describing biological actions of chemokines during various pathological conditions such as infection, preterm labor and preterm rupture of membranes suggest that these compounds play important roles in other gestational events such as cervical dilation and myometrial contractions. Even though we do not know the totality of biochemical signals that integrate the molecular dialogue between leukocytes and the various gestational tissues, it is becoming increasingly evident that this microenvironment is characterized, at least in part, by the differential expression and secretion of chemokines that induce selective trafficking of leukocyte subsets to the fetal membranes. Therefore, chemokines should be considered as important regulatory molecules with the ability to initiate the events that characterize normal and pathological labor.

A prolific allele named FecL^L is known to segregate in the meat breed of the French Lacaune sheep on the basis of ovulation rate record. To gain more knowledge about the physiological effects of FecL^L, ewes homozygous for FecL^L (L/L) were compared to wild-type ewes ( / ) for ovarian phenotype and reproductive endocrine profiles. At the ovarian level, the increased ovulation rate in L/L ewes was associated with an increased number of antral follicles of greater than 3 mm and with preovulatory follicles being, on average, 1 mm smaller. Intrafollicular estradiol and testosterone concentrations were not significantly different between the two genotypes. In contrast, L/L large follicles (≥6 mm) had lower intrafollicular progesterone concentration. At the molecular level, expressions of ovarian markers, such as CYP19A1, CYP11A1, CYP17A1, LHR, and INHA, were not significantly different between the two genotypes. In contrast, FSHR and STAR mRNA levels increased in granulosa cells from L/L ewes. Plasma concentrations of estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone measured across a synchronized estrous cycle revealed a significant increase in estradiol levels during the follicular phase, a precocious LH surge, and an increase in progesterone level during the luteal phase of L/L ewes compared to / ewes. Circulating concentrations of FSH were not different between the two genotypes. The precocious LH surge was associated with an increase in frequency of LH pulsatility during the follicular phase. At the pituitary level, mRNA levels for LHB, FSHB, GNRHR, and ESR1 were not significantly different between the two genotypes. In contrast, ESR2 mRNA expression was decreased in L/L ewes compared to / ewes. Based on ovarian phenotype and endocrine profiles, these findings suggest that the mutation in the FecL gene affects ovarian function in a different way compared to other known prolific mutations affecting the bone morphogenetic protein signaling system in the ovine species.

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development.

During pregnancy, the integrity of the mammary gland epithelium must be maintained by the junctional nexus formed by gap, adherens, and tight junctions for proper alveologenesis. We have previously shown that mammary glands from Gja1^Jrt/ mice, harboring a mutation in the Gja1 gene encoding connexin (Cx) 43, have reduced Cx43 gap junction function and are unable to release milk to the pups. We hypothesized that low levels of Cx43 in the mammary gland may also affect other mammary gland connexins, tight and adherens junction proteins, and alveolar architecture, thus contributing to the lactation defect. Surprisingly, the levels and/or localization of Cx26 (Gjb2), Cx30 (Gjb6), Cx32 (Gjb1), E-cadherin, P-cadherin, beta-catenin, claudin-1, and occludin were all unaffected by the decreased levels of Cx43 at parturition, suggesting no cross-talk to other connexins or junctional proteins in mutant mice mammary glands. Consistently, in mammary gland myoepithelial primary cultures or in Normal Rat Kidney (NRK) cells, tight and adherens junction protein levels and localization were unaffected by the expression of Cx43 mutants. However, whereas the architecture of the alveolar and ductal structures appeared to be normal in mutant mice, the presence of milk and cytoplasmic lipid droplets were frequently observed within the epithelium itself, suggesting a defect in the secretory process. Overall, these results suggest that proteins associated with tight and adherens junctions are unaffected by greatly reduced Cx43 gap junctions in the mammary gland epithelium, and they further suggest that the mammary gland defect in Cx43 mutant mice may extend to impaired milk secretion.

To increase the manipulation efficiency and storage capability of vitrified human embryonic stem cells, a new bulk vitrification method was established using transformed cryovials. This method vitrified a large number of cell clumps, as opposed to those cryopreserved by a slow-freezing method with conventional cryovials at one time (round). After warming, vitrified human embryonic stem cells exhibited a much higher survival rate than the slow-freezing cells. The vitrified stem cells continued to express markers of pluripotency and formed teratomas in mice with severe combined immunodeficiency, confirming the pluripotency of vitrified-warmed human embryonic stem cell clumps. The new bulk vitrification method is superior to and more practical than the open pulled straw vitrification method and the slow-freezing method for the cryopreservation of human embryonic stem cells.

Cathepsins (CTSB and CTSL1) and their inhibitor, cystatin C (CST3), remodel uterine endometrium and placenta for transport of gases, micronutrients, and macromolecules essential for development and growth of the conceptus (embryo/fetus and placental membranes). We examined the temporal/spatial control of expression for CTSB, CTSL1, and CST3 mRNAs in endometria and placentae of pigs using three developmental models: 1) pigs were hysterectomized during the estrous cycle or pregnancy; 2) cyclic pigs were injected with estrogen to induce pseudopregnancy and were hysterectomized; and 3) pigs were ovariectomized, injected with progesterone, and hysterectomized. The abundance of CTSB, CTSL1, and CST3 mRNAs increased in endometrial epithelia during pregnancy and in response to exogenous progesterone but not estrogen. CST3 was also expressed in cells scattered within the stratum compactum stroma. Progesterone decreased epithelial but increased stromal compartment expression of CST3. CTSB increased in all chorionic epithelia, but CTSL1 was limited to chorionic epithelia that form areolae to absorb secretions from uterine glands. Based on the placental and endometrial distribution of CTSL1, we examined expression in the neonatal enterocytes known to transport immunoglobulins from colostrum. CTSL1 was also expressed in enterocytes of intestine from neonatal piglets. Therefore, CTSL1 is expressed by endometrial epithelia, placental areolae, and neonatal intestine, and it may function in the transport of macromolecules across these epithelia. Our results support the idea that reciprocal interactions between CSTL1, CTSB, and CST3 may be required to remodel endometrial and placental tissues for close apposition between maternal and fetal vasculatures and to facilitate transplacental transport of gases, micronutrients (amino acids, glucose), and macromolecules (proteins). Cysteine proteases and their inhibitors may also specifically modify proteins for successful utilization and fluid-phase transport across uterine, placental, and neonatal gut epithelia.

WNTs are secreted extracellular signaling molecules that transduce their signals by binding to G protein-coupled receptors of the frizzled (FZD) family. They control diverse developmental processes, such as cell fate specification, cell proliferation, cell differentiation, and apoptosis. Although WNT signaling has been shown to be essential for development of the ovary, its mechanistic role in folliculogenesis within the adult ovary has not been studied extensively. Therefore, the objective of this study was to investigate the regulation and function of WNT2 signaling in mouse granulosa cells. Immunostaining identified WNT2 as being expressed in granulosa cells throughout folliculogenesis, but with varying signal strength: in sequential sections, WNT2 immunoreactivity was strongest in healthy antral follicles but weak in atretic follicles. Knockdown of WNT2 expression using transfected short interfering RNA decreased DNA synthesis in granulosa cells, whereas WNT2 overexpression using a recombinant viral vector enhanced it. WNT2 knockdown led to accumulation of glycogen synthase kinase-3beta (GSK3B) in the cytoplasm but reduced the expression of beta-catenin. Conversely, WNT2 overexpression reduced the expression of GSK3B in the cytoplasm and induced beta-catenin translocation from the membrane into the nucleus. Beta-catenin knockdown also inhibited DNA synthesis in granulosa cells and neutralized the effect of WNT2 overexpression. WNT2/beta-catenin signaling had a slight effect on the apoptosis of granulosa cells. Taken together, the data indicate that WNT2 regulates beta-catenin localization in granulosa cells, and WNT2/beta-catenin signaling contributes to regulating their proliferation.

Because sphingosine 1-phosphate (S1P) is a potent stimulator of angiogenesis, we hypothesized that the S1P pathway is activated to stimulate endometrial/placental angiogenesis during pregnancy. We initially localized S1P signaling pathway members in the gravid and nongravid uterine horns of unilaterally pregnant ewes. Sphingosine kinase-1 expression was greater in gravid compared to nongravid horns. In situ hybridization revealed elevated expression of sphingosine 1-phosphate phosphatase (SGPP1) in gravid interplacentomal endometrial stroma on Days 20 and 40 compared to the nongravid uterine horn, but expression increased in endometrium of the nongravid uterine horn between Days 40 and 120. SGPP1 expression increased in placentomes late in gestation. Sphingosine 1-phosphate lyase mRNA was modestly expressed at Day 20 and then decreased. In contrast, sphingosine 1-phosphate receptor 1 (S1PR1) mRNA increased in endometrium and caruncular stroma of the gravid uterine horn. Treatment with FTY720 and VPC23019, S1P receptor antagonists, blocked human and ovine endothelial cell invasion using an in vitro model of sprouting angiogenesis. Knockdown of S1PR1 with siRNA reduced invasion responses as well. We previously reported that delta-like 4 (DLL4) and A disintegrin and metalloproteinase with thrombospondin-like repeats 1 (ADAMTS1) participate in endothelial cell invasion stimulated by S1P and growth factors in vitro, and thus investigated whether their expression correlated with areas undergoing angiogenesis in vivo. DLL4 expression was similar to S1PR1, while ADAMTS1 mRNA was expressed by endometria of both nongravid and gravid horns, as well as conceptus and placentomes. These results establish that S1P signaling pathway members and S1P- and growth factor-regulated genes are prominent in uterine and placental tissue and in some cases are correlated with areas undergoing angiogenesis. Thus, S1P signaling may be crucial for proper fetal-placental development.

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a “testicular xenograft.” We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels.

The production of xenogenic gametes from large-bodied, commercially important marine fish species in closely related smaller host fish species with short generation times may enable rapid and simple seed production of the target species. As a first step toward this goal, we assessed the suitability of chub mackerel, Scomber japonicus, as a small-bodied recipient species for xenogenic spermatogonial transplantation. Histological observation of the early gonadal development of chub mackerel larvae and transplantation of fluorescent-labeled spermatogonia from Nibe croaker, Nibea mitsukurii, revealed that 5.3-mm chub mackerel larvae were suitable recipients for successful transplantation. Intraperitoneally transplanted xenogenic spermatogonia efficiently colonized the gonads of these recipient larvae, and donor-derived Nibe croaker germ cells proliferated rapidly soon after colonization. Moreover, gonadal soma-derived growth factor (gsdf) mRNA, a gonadal somatic cell marker, was expressed in recipient-derived cells surrounding the incorporated donor-derived germ cells, suggesting that donor-derived germ cells had settled at an appropriate location in the recipient gonad. Our data show that xenogenic spermatogonial transplantation was successful in chub mackerel and that the somatic microenvironment of the chub mackerel gonad can support the colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells derived from a donor species of a different taxonomic family.

Reactive oxygen species may cause oxidative damage in the placenta, yet some mechanisms must exist to reduce or prevent such damage. We investigated whether oxidative injury to placental endothelial cells is inhibited by activation of antioxidant enzymes by paracrine factors secreted by human placental multipotent mesenchymal stromal cells (hPMSC). hPMSC-conditioned medium and umbilical endothelial cells were assayed for cytokines and cytokine receptor expression by immunoassay and real-time PCR. Endothelial cell survival was evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and caspase 3 activity assay. tert-Butyl hydroperoxide was used to induce oxidative injury in endothelial cells, with fluorescent microscopy and flow cytometry used to detect intracellular peroxides and cell apoptosis. Western blot, real-time PCR, STAT3 DNA-binding activity assay, and STAT3 siRNA were used to assess endothelial cell antioxidant enzymes. hPMSC-conditioned medium supported endothelial cell survival and reduced endothelial cell intracellular peroxides and apoptosis. hPMSCs expressed the transcripts of the interleukin (IL) 6 cytokine family, including IL6 and leukemia-inhibitory factor. hPMSC-conditioned medium activated STAT3 expression in endothelial cells, which was inhibited by neutralizing antibody to interleukin 6 signal transducer (IL6ST) but not to IL6 or leukemia-inhibitory factor. STAT3 siRNA or manganese superoxide dismutase (SOD2) siRNA transfected into endothelial cells inhibited the antiapoptotic effect of conditioned medium. SOD2 was significantly upregulated in endothelial cells by conditioned medium via STAT3 activation that, in turn, was inhibited by IL6ST-neutralizing antibody or STAT3 siRNA. Paracrine factors secreted by hPMSCs support endothelial cell survival. STAT3 activation and SOD2 production protect against oxidative stress-induced endothelial cell damage.

Maternal exposure to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a variety of defects in compaction-stage embryos, including monopolar spindle formation, errors in chromosome segregation, and fragmentation resulting from aberrant cytokinesis. In this study, we investigated the possibility that a failure in centrosome duplication, separation, or positioning within blastomeres might underlie the observed effects of TCDD on early embryos. The subcellular localization of the centrosomal marker TUBG1 was analyzed in preimplantation embryos collected from female rats exposed to either chronic (50 ng kg⁻¹ wk⁻¹ for 3 wk) or acute (50 ng/kg or 1 μg/kg at proestrus) doses of TCDD. In treated embryos, interphase TUBG1 foci were more abundant and cortically displaced when compared to those in controls. At prophase, some blastomeres exhibited a single large perinuclear TUBG1 aggregate, suggesting a failure in centrosome duplication or separation. Furthermore, the presence of monopolar spindles at metaphase was confirmed by the localization of TUBG1 to the single spindle pole. Therefore, the misregulation of centrosome number and localization, as indicated by TUBG1 staining, may contribute to errors in chromosome segregation and cytokinesis in embryos following maternal TCDD exposure.

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1–2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle.

The essential role of endometrial prostaglandin F2 alpha (PTGF) for induction of the corpus luteum (CL) regression is well documented in the cow. However, the acute effects of PTGF on known local luteotropic factors (oxytocin [OXT] and its receptor, insulin-like growth factor [IGF] 1, and progesterone and its receptor), the principal angiogenic factor vascular endothelial growth factor (VEGF) A and the capillary destabilization factor angiopoietin (ANGPT) 2 were not thoroughly studied in detail. The aim of this study was therefore to evaluate the tissue concentration of these factors during PTGF induced luteolysis. In addition the mRNA expression of progesterone receptor (PGR), OXT receptor (OXTR), IGF1, IGFBP1, ANGPT1, and ANGPT2 was determined at different times after PTGF treatment. Cows (n = 5 per group) in the mid-luteal phase (Days 8–12, control group) were injected with the PTGF analog (cloprostenol), and CL were collected by transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h after injection. The mRNA expression was analyzed by quantitative real-time PCR, and the protein concentration was evaluated by enzyme immunoassay or radioimmunoassay. Progesterone concentrations, as well as mRNA expression of PGR, in CL tissue were significantly down-regulated by 12 h after PTGF. Tissue OXT peptide and OXTR mRNA decreased significantly after 2 h, followed by a continuous decrease of OXT mRNA. IGF1 and VEGFA protein already decreased after 0.5 h. By contrast, the IGFBP1 mRNA was up-regulated significantly after 2 h to a high plateau. ANGPT2 protein and mRNA significantly increased during the first 2 h, followed by a steep decrease after 4 h. The acute decrease of local luteotropic activity and acute changes of ANGPT2 and VEGFA suggest that modulation of vascular stability may be a key component in the cascade of events leading to functional luteolysis.

Male mice deficient in ESR1 (ERalpha) (Esr1KO mice) are infertile, and sperm recovered from the cauda epididymis exhibit reduced motility and fail to fertilize eggs in vitro. These effects on sperm appear to result from defective epididymal function and not a direct effect on spermatogenesis, as Esr1KO germ cells transplanted into wild-type testes yield normal offspring. We hypothesized that the previously described defect in efferent duct fluid reabsorption would lead to alterations in the epididymal fluid milieu, which would negatively impact sperm function. Analysis of the epididymal fluid revealed that the Esr1KO maintains a higher luminal pH throughout the epididymis, confirming an inability of the efferent ducts and/or epididymis to properly acidify the luminal contents. Subsequent studies showed that these abnormalities were not the result of global defects in epididymal function since protein secretion by the Esr1KO epididymis appeared normal as judged by SDS-PAGE of total secreted proteins and by immunoblotting of candidate secreted proteins. To gain insight into the basis of the aberrant fluid homeostasis in the Esr1KO epididymis, the expression of several enzymes and transporters known to be involved in acid/base regulation were analyzed. The levels of SLC9A3 (NHE3) as well as carbonic anhydrase XIV and SLC4A4 (NBC1) were all reduced in the proximal portion of the Esr1KO epididymis, while other components appeared unaffected, including other ion transporters and ATP6V0A1 (V-ATPase). The altered luminal milieu of the Esr1KO epididymis was shown to lead to a corresponding increase in the intracellular pH of Esr1KO sperm, relative to sperm from control animals. Since pH and bicarbonate ions are critical regulators of sperm cAMP levels and motility, we attempted to bypass the abnormal luminal and intracellular environment by supplementing sperm with exogenous cAMP. This treatment rescued all defective motility parameters, as assayed by CASA, further showing that motility defects are not intrinsic to the sperm but, rather, result from the abnormal epididymal milieu.

Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

CD82 is recognized as a wide-spectrum tumor metastasis suppressor that inhibits cancer cell motility and invasiveness. At the human maternal-fetal interface, the decidua is believed to effectively limit the inappropriate invasion of trophoblasts. Here we have found the transcription and translation of CD82 in decidual stromal cells (DSCs), whereas trophoblast cells do not express CD82. The in-cell Western analysis reveals attenuation of CD82 translation in DSCs by human chorionic gonadotropin (hCG), but not by estrogen or progesterone. It is demonstrated that silencing of CD82 by RNA interference increases integrinβ1, decreases TIMP1 expression in DSCs, and promotes the invasion of the first-trimester human trophoblasts in the coculture. Moreover, U0126, or anti-integrinβ1 neutralizing antibody, reverses the decreased TIMP1 expression and the increased invasiveness of trophoblast cells, and the antibody also inhibits the MAPK3/1 phosphorylation induced by CD82 silence. After transfection with CD82, the invasive index of BeWo cells decreases significantly with TIMP1 increase. The results above indicate that the DSCs-expressed CD82 up-regulates the expression of TIMP1 in an autocrine manner and inhibits the invasiveness of human first-trimester trophoblast cells partly through the integrinβ1/MAPK/MAPK3/1 signaling pathway. Furthermore, we have found that the mRNA and protein level of CD82 in decidua of the miscarriage is significantly higher than that of the normal early pregnancy, which implies that the abnormal higher CD82 expression in decidua restricts appropriate invasion of trophoblasts that leads to early pregnancy wastage.

The transforming growth factor beta superfamily ligand activin A controls juvenile testis growth by stimulating Sertoli cell proliferation. Testicular levels are highest in the first postnatal week, when Sertoli cells are proliferating and spermatogonial stem cells first form. Levels decrease sharply as Sertoli cell proliferation ceases and spermatogenic differentiation begins. We hypothesized that changing activin levels also affect germ cell maturation. We detected an acute and developmentally regulated impact of activin on Kit mRNA in cocultures of Sertoli cells and germ cells from Day 8, but not Day 4, mice. Both stereological and flow cytometry analyses identified an elevated spermatogonium:Sertoli cell ratio in Day 7 testes from Inhba^BK/BK mice, which have decreased bioactive activin, and the germ cell markers Sycp3, Dazl, and Ccnd3 were significantly elevated in Inhba^BK/BK mice. The flow cytometry measurements demonstrated that surface KIT protein is significantly higher in Day 7 Inhba^BK/BK germ cells than in wild-type littermates. By Day 14, the germ cell:Sertoli cell ratio did not differ between genotypes, but the transition of type A spermatogonia into spermatocytes was altered in Inhba^BK/BK testes. We conclude that regulated activin signaling not only controls Sertoli cell proliferation, as previously described, but also influences the in vivo progression of germ cell maturation in the juvenile testis at the onset of spermatogenesis.

Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihh acts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihh action during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgr^tm2(cre)Lyd (PR^cre) mouse model [Pgr^tm2(cre)Lyd Gt(ROSA)26Sor^{tm1(Smo/EYFP)Amc} (PR^cre/ SmoM2 )]. Female PR^cre/ SmoM2 mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PR^cre/ SmoM2 uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PR^cre/ SmoM2 uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PR^cre/ SmoM2 increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.

Poly(ADP-ribosyl)ation is a posttranslational protein modification carried out by a family of enzymes referred to as poly(ADP-ribose) polymerases (PARPs). It has been proposed that the broad nuclear distribution of PARPs may allow them to modulate gene expression in addition to their more accepted role as DNA repair mediators. The role of poly(ADP-ribosyl)ation during oogenesis and folliculogenesis is unknown. Here we found that when 3- to 4-wk-old mice were injected with 5-amninoisoquinolinone, a water soluble inhibitor of poly(ADP-ribosyl)ation, it leads to considerably increased oocyte numbers and a dramatic increase in primordial follicle numbers. Furthermore, we show that inhibition of poly(ADP-ribosyl)ation leads to an increased expression of specific genes and pathways in mouse ovaries, in particular, transforming growth factor superfamily members. Our results demonstrate that poly(ADP-ribosyl)ation, is important in oogenesis and folliculogenesis, and it may have a differential role in regulating gene expression, DNA repair, and apoptosis. The novel function of poly(ADP-ribosyl)ation in oogenesis and folliculogenesis sheds light on the alternative role that DNA repair mediators may play in cellular development and differentiation.

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.