Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.

The requirement for vitamin A in reproduction and development was first determined from studies of nutritional deficiencies. Subsequent research has shown that embryonic development and both male and female reproduction are modulated by retinoic acid (RA), the active form of vitamin A. Because RA is active in multiple developmental systems, its synthesis, transport, and degradation are tightly regulated in different tissues. A growing body of evidence implicates RA as a requirement for the initiation of meiosis in both male and female mammals, resulting in a mechanistic model involving the interplay of RA, RA synthesis enzymes, RA receptors, and degradative cytochrome P450 enzymes in this system. Recently, that model has been challenged, prompting a review of the established paradigm. While it remains possible that additional molecules may be involved in regulating entry into meiosis, the weight of evidence supporting a key role for RA is incontrovertible.

It is well established that syncytium formation involves the fusion of mononucleated trophoblasts into a multinucleated structure and the secretion of hormonal factors, such as human chorionic gonadotropin (hCG). These morphological and biochemical changes are regulated by a plethora of ligands, which upon binding to specific receptors trigger the activation of many signaling pathways, such as janus kinase/signal transducer and activator of transcription (JAK/STAT) and the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinases 1 and 2 (MAPK3/1). We used the forskolin-induced syncytialization of trophoblastlike BeWo cells to characterize at the cellular level the effect mediated by leukemia inhibitory factor (LIF) on trophoblast differentiation and to describe its action at the molecular level. Forskolin induces both hCG secretion and BeWo cell syncytial fusion. Although LIF had no effect on the undifferentiated state of the cells, the cytokine generated a strong reduction in forskolin-induced hCG release. In contrast to its effect on hCG secretion, LIF exerts a synergistic effect toward forskolin-induced fusion. LIF reduced hormonal production through a STAT1- and STAT3-dependent mechanism, whereas MAPK3/1 was not involved in this process. However, both types of signaling molecules were required to mediate the action of LIF in forskolin-induced cell fusion. These data provide novel insights into the regulation of trophoblast cell differentiation by LIF and describe for the first time the molecular mechanism underlying the effect of the cytokine.

The ex situ population of the Przewalski's horse (Equus ferus przewalskii) is not self-sustaining (20% foaling rate), and the demography is skewed toward aging individuals with low gene diversity. We designed the present study to gain a better understanding of the reproductive biology of the Przewalski's mare and to determine whether age and gene diversity influenced reproductive function. Urine samples were collected 3–7 days/wk from 19 mares from May to September, and ultrasound examinations of follicular structures were performed 3 days/wk for 5 wk from May through July in nine individuals. A high proportion of mares exhibited abnormal (endocrine, 5 [26.3%] of 19; follicular, 2 [22.2%] of 9) or acyclic (endocrine, 4 [21.1%] of 19; follicular, 3 [33.3%] of 9) reproductive patterns. In four cyclic mares, estrous cycle length was 25.1 ± 1.2 days, with 12.2 ± 0.9 days of diestrus. Follicles in cyclic mares grew 1.2 ± 0.6 mm per day and ovulated after reaching 40.4 ± 8.9 mm. Mares with a high coefficient of inbreeding excreted reduced levels of mean urinary estrogens (r² = 0.476, P < 0.05), but age had no significant impact on reproductive patterns in this population. Overall, these data suggest that long-term genetic management of this population is necessary to maintain reproductive fitness.

Men and women differ in their susceptibility to sexually transmittable infections (STIs) such as human immunodeficiency virus (HIV). However, a paucity of published information regarding the tissue structure of the human genital tract has limited our understanding of these gender differences. We collected cervical, vaginal, and penile tissues from human adult donors. Tissues were prepared with hematoxylin and eosin stains or immunofluorescence labeling of epithelial cell proteins and were analyzed for structural characteristics. Rhesus macaque genital tissues were evaluated to assess the use of this model for HIV/simian immunodeficiency virus transmission events. We found the stratified squamous epithelia of the male and female genital tract shared many similarities and important distinctions. Expression of E-cadherins, desmogleins 1/2, and involucrin was seen in all squamous epithelia, though expression patterns were heterogeneous. Filaggrin and a true cornified layer were markedly absent in female tissues but were clearly seen in all male epithelia. Desmogleins 1/2 were more consistent in the outermost strata of female squamous genital epithelia. Macaque tissues were similar to their respective human tissues. These initial observations highlight how male and female genital epithelia resemble and differ from one another. Further information regarding tissue structural characteristics will help to understand how STIs traverse these barriers to cause infection. This knowledge will be essential in future HIV pathogenesis, transmission, and prevention studies.

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.

During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.

Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.

Human endometrium regenerates on a cyclic basis from candidate stem/progenitors whose genetic programs are yet to be determined. A subpopulation of endometrial stromal cells, displaying key properties of mesenchymal stem cells (MSCs), has been characterized. The endometrial MSC (eMSC) is likely the precursor of the endometrial stromal fibroblast. The goal of this study was to determine the transcriptome and signaling pathways in the eMSC to understand its functional phenotype. Endometrial stromal cells from oocyte donors (n = 20) and patients undergoing benign gynecologic surgery (n = 7) were fluorescence-activated cell sorted into MCAM (CD146) /PDGFRB (eMSC), MCAM (CD146)⁻/PDGFRB (fibroblast), and MCAM (CD146) /PDGFRB⁻ (endothelial) populations. The eMSC population contained clonogenic cells with a mesenchymal phenotype differentiating into adipocytes when cultured in adipogenic medium. Gene expression profiling using Affymetrix Human Gene 1.0 ST arrays revealed 762 and 1518 significantly differentially expressed genes in eMSCs vs. stromal fibroblasts and eMSCs vs. endothelial cells, respectively. By principal component and hierarchical clustering analyses, eMSCs clustered with fibroblasts and distinctly from endothelial cells. Endometrial MSCs expressed pericyte markers and were localized by immunofluorescence to the perivascular space of endometrial small vessels. Endometrial MSCs also expressed genes involved in angiogenesis/vasculogenesis, steroid hormone/hypoxia responses, inflammation, immunomodulation, cell communication, and proteolysis/inhibition, and exhibited increased Notch, TGFB, IGF, Hedgehog, and G-protein-coupled receptor signaling pathways, characteristic of adult tissue MSC self-renewal and multipotency. Overall, the data support the eMSC as a clonogenic, multipotent pericyte that displays pathways of self-renewal and lineage specification, the potential to respond to conditions during endometrial desquamation and regeneration, and a genetic program predictive of its differentiated lineage, the stromal fibroblast.

Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.

Dynamic alterations in chromatin configuration occur in mammalian oocytogenesis. Based on chromatin configuration patterns, fully grown oocytes are classified into two types. One is surrounded nucleolus (SN)-type and the other is nonsurrounded nucleolus (NSN)-type oocytes. Although chromatin condensation during the transition from NSN- to SN-type oocytes is a prerequisite for normal early embryonic development, the molecular mechanisms remain unclear. In this study, we analyzed the role of DPPA3 (also known as PGC7/Stella) in this transition using Dppa3-null oocytes. The NSN-to-SN transition was significantly impaired, and transcriptional repression was incomplete in the Dppa3-null oocytes. Additionally, we revealed that prior transcriptional repression was necessary for the NSN-to-SN transition. These findings demonstrate that DPPA3 is an essential factor for the production of functional oocytes through transcriptional repression and chromatin condensation.

Human eggs are highly aneuploid, with female age being the only known risk factor. Here this aging phenomenon was further studied in Swiss CD1 mice aged between 1 and 15 mo. The mean number of eggs ± SEM recovered from mice following superovulation peaked at 22.5 ± 3.8 eggs/oviduct in 3-mo-old females, decreasing markedly between 6 and 9 mo old, and was only 2.1 ± 0.2 eggs/oviduct by 15 mo. Measurement of aneuploidy in these eggs revealed a low rate, ∼3–4%, in mice aged 1 and 3 mo, rising to 12.5% by 9 mo old and to 37.5% at 12 mo. Fifteen-month-old mice had the highest rate of aneuploidy, peaking at 60%. The in situ chromosome counting technique used here allowed us to measure with accuracy the distance between the kinetochores in the sister chromatids of the eggs analyzed for aneuploidy. We observed that this distance increased in eggs from older females, from 0.38 ± 0.01 μm at 1 mo old to 0.82 ± 0.03 μm by 15 mo. Furthermore, in 3- to 12-mo-old females, aneuploid eggs had significantly larger interkinetochore distances than euploid eggs from the same age, and measurements were similar to eggs from the oldest mice. However, the association between aneuploidy and interkinetochore distance was not observed at the oldest, 15-mo age, despite such measurements being maximal. We conclude that in aging CD1 mice, a reduction in the ovulated egg number precedes a rise in aneuploidy and, furthermore, except at very advanced ages, increased interkinetochore distance is associated with aneuploidy.

The development of a new male contraceptive requires a transition from animal model to human and an understanding of the mechanisms involved in the target's inhibition of human spermatozoan fertility. We now report that semenogelin (SEMG1) and anti-EPPIN antibodies to a defined target site of 21 amino acids on the C terminal of EPPIN cause the loss of intracellular calcium, as measured by Fluo-4. The loss of intracellular calcium explains our previous observations of an initial loss of progressive motility and eventually the complete loss of motility when spermatozoa are treated with SEMG1 or anti-EPPIN antibodies. Thimerosal can rescue the effects of SEMG1 on motility, implying that internal stores of calcium are not depleted. Additionally, SEMG1 treatment of spermatozoa decreases the intracellular pH, and motility can be rescued by ammonium chloride. The results of this study demonstrate that EPPIN controls sperm motility in the ejaculate by binding SEMG1, resulting in the loss of calcium, most likely through a disturbance of internal pH and an inhibition of uptake mechanisms. However, the exact steps through which the EPPIN-SEMG1 complex exerts its effect on internal calcium levels are unknown. Anti-EPPIN antibodies can substitute for SEMG1, and, therefore, small-molecular weight compounds that mimic anti-EPPIN binding should be able to substitute for SEMG1, providing the basis for a nonantibody, nonhormonal male contraceptive.

Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%–99% decrease in basal transepithelial electrical resistance (R_TE, a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R_TE significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.

In sheep, the seasonal patterns of reproductive activity are driven primarily by the annual photoperiodic cycle, but can also respond to other environmental factors, such as nutrition, yet little is known about the mechanisms underlying this interaction. This study was designed to define the interaction between photoperiodic and nutritional cues on seasonal patterns of ovarian activity, and to determine if there is a central interaction between these cues. Groups of Ile-de-France ewes were maintained in two nutritional states (restricted and well fed) under a simulated annual photoperiod of 8–16 h of light per day over two breeding seasons. At the end of the first breeding season, half of the animals of each group were ovariectomized (OVX) and fitted subcutaneously with estradiol implants. Low nutritional status shortened the season of ovarian activity, determined from the pattern of progesterone concentrations, by modifying the timing of seasonal transitions between periods of ovarian activity and anestrus. The same results were observed for the seasonal rhythm of neuroendocrine activity, assessed in the OVX ewes, from the pattern of luteinizing hormone concentrations. These results were then confirmed for neuroendocrine activity induced by a photoperiodic treatment. We conclude that nutrition centrally modulates the interpretation of photoperiod to affect seasonal reproductive transitions. The mechanisms of this interaction are discussed in the paper.

The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture of cells. Functional interactions exist between peripheral T cells and luteal cells in vitro; however, the precise role of resident T cells (RTC) remains unknown. The goals of the present study were to isolate RTC from within the CL and determine if alteration of luteal function resulted in changes in RTC phenotypes. Functional lymphocyte phenotypes identified in the bovine CL by using quantitative flow cytometric analysis were clearly different from those in the peripheral circulation. The proportion of CD8 RTC was greater than CD4 RTC. These proportions were opposite in peripheral blood. The proportion of γδ lymphocytes was not different in the CL compared to that in peripheral blood nor was it altered during luteal regression. There was a significant increase in CD8αα and γδ CD8αα RTC during luteal regression. The proportion of FOXP3 lymphocytes in the CL was greater than that isolated from peripheral blood, and this proportion of lymphocytes was dramatically reduced by induction of luteolysis. Within the CL of early pregnancy, there was an increase in the CD8αβ and γδ CD8αβ populations compared to those in the CL of nonpregnant animals. Based on these data, we concluded that the functional state of the CL creates a microenvironment that regulates the recruitment of or differentiation into specific lymphocyte types. Understanding the interactions between steroidogenic cells and ovarian lymphocytes within CL will not only enhance understanding of reproductive function but may provide vital clues to lymphocyte regulation within tissues.

The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally, and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.

In most oviparous animal species, oocyte growth occurs via the uptake of plasma egg yolk precursors, predominantly vitellogenins (Vtg). These glycolipoproteins are members of the large lipid transfer protein superfamily and key players in reproduction. While the vertebrate liver has been demonstrated to synthesize large amounts of Vtg, mostly under 17beta-estradiol control, the ability of other tissues to express significant amounts of Vtg has not been conclusively demonstrated. RT-PCR revealed vtg1 transcripts in female zebrafish and rainbow trout white adipose tissue (WAT). It was also found to coexpress mtp, known to perform the intracellular lipidation of Vtg prior to secretion. The liver and pancreas markers apobb2 and ins, or ela2, respectively, were not expressed in adipocytes. Whole-mount in situ hybridization and in situ RT-PCR tests of histological sections revealed vtg1 signal in adipocytes, whereas no signal was detected in infiltrated pancreatic islets. Transcript expression of vtg1 was induced in WAT of 17beta-estradiol-treated males, and the transcript and corresponding protein were detected in the thin rim of cytoplasm surrounding the adipocyte. Real-time quantitative RT-PCR showed that rainbow trout perivisceral WAT vtg1 transcript levels were high during early compared to late vitellogenesis. Taking normalized mRNA levels and tissue somatic index into account, vtg1 transcript levels at the beginning of oocyte yolk deposition were approximately 45 times lower in WAT than in liver, and these levels were not correlated to plasma Vtg and 17beta-estradiol concentrations. These findings suggest that WAT Vtg is implicated in providing components to the ovary during the early stages of vitellogenesis.

Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3′ untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.

Both the systemic and the uteroplacental renin-angiotensin system (RAS) display dramatic changes during pregnancy. However, whether gestational protein insufficiency affects the expressions of RAS in the placenta remains unknown. In this study, we hypothesized that the expression of Ace2 in the placental labyrinth was reduced by maternal protein restriction. Pregnant Sprague-Dawley rats were fed a normal diet or a low-protein diet (LP) from Day 1 of pregnancy until they were killed at Day 14 or Day 18. The labyrinth zone (LZ) of the placenta was then dissected and snap frozen for expression analysis by quantitative real-time PCR of Ace, Ace2, Agtr1a, Agtr1b, and Agtr2. Formalin-fixed placentas were used for immunohistochemical analysis on ACE and ACE2 proteins. The findings include 1) the expression of Ace2 in rat LZ was reduced by maternal protein restriction in late pregnancy; 2) ACE protein was mainly present in syncytiotrophoblasts, whereas ACE2 protein was found predominantly in fetal mesenchymal tissue and fetal capillaries; 3) Agtr1a was predominant in the rat LZ, and its mRNA levels, but not protein levels, were reduced by LP; 4) expressions of Ace, Ace2, and Agtr1a in the rat LZ and their response to LP occurred in a gender-dependent manner. These results may indicate that a reduced expression of Ace2 and perhaps an associated reduction in angiotensin (1–7) production in the placenta by maternal protein restriction may be responsible for fetal growth restriction and associated programming of adulthood hypertension.

Preterm delivery is the leading cause of perinatal mortality and morbidity. Current tocolytics target myometrial contractions, a late step in the labor cascade. Identifying earlier events in parturition may lead to more effective therapeutic strategies. We hypothesized that inflammatory events in decidua (the maternal-fetal interface), characterized by leucocyte infiltration, are an early event during term and preterm labor (PTL). Leucocyte abundance in decidua of human pregnancies was quantified following term labor and PTL (idiopathic and infection associated), in conjunction with investigation of temporal inflammatory events in rat uterus during the perilabor period and in PTL induced by mifepristone. In human decidua, macrophage numbers were 4-fold higher in term labor (P < 0.01) and 2.5-fold higher in non-infection-associated PTL (P < 0.05) than in term nonlaboring samples. Neutrophil abundance was unchanged with labor but elevated in PTL with infection (5- to 53-fold increase; P < 0.01). T and NK cells were more abundant in idiopathic PTL than TL (P < 0.05). In rat, decidual macrophage infiltration increased 4.5-fold 12 h prior to labor and remained elevated during labor and early postpartum (P < 0.01). Decidual infiltration preceded that of the myometrium and was 4-fold higher (P < 0.01). In rat PTL, decidual macrophage numbers were also elevated (P < 0.01) and exceeded those of the myometrium (P < 0.05). These studies show for the first time that leucocytes infiltrate decidua during labor at term and preterm, supporting a role for leucocyte-derived inflammatory mediators in decidual activation. In the rat, this occurred prior to labor, suggesting it is an early event during parturition and thus a potential target for intervention.

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

It has been proposed that four members of the aquaporin family (AQPs 1, 3, 8, and 9) are involved in the control of amniotic fluid (AF) homeostasis, as illustrated by their differential expression patterns in normal and pathological human term fetal membranes. However, there are no data available to date on their ontogeny throughout pregnancy. Our objective was to determine spatiotemporal expression profiles of the mRNA and proteins of all 13 members of this transmembrane channel family. For this purpose, we used healthy fetal membranes from the first, second, and third trimesters of pregnancy. Total mRNA and proteins were extracted from total membranes and from separated amnion and chorion. Quantitative PCR, Western blot, and immunohistochemistry experiments were carried out to determine the presence of AQPs and to quantify their spatiotemporal expression patterns throughout pregnancy. The WISH cell line was tested to propose a cellular model for the role of AQPs in the amnion compartment. AQP11 expression was established in amniotic membranes at term. Aquaporins 1, 3, 8, 9, and 11 mRNA and proteins were present in amnion and chorion throughout human gestation. Each AQP has a time-specific expression pattern, with AQP1 presenting the highest variation in terms of mRNA and protein levels. The WISH cell line also expressed the same five AQPs. Taken together, these results indicate that AQPs are expressed and potentially involved in the regulation of AF homeostasis throughout pregnancy. This also clearly supports the hypothesis that abnormal expression could occur at any time during pregnancy, ultimately leading to obstetrical pathologies such as polyhydramnios or oligohydramnios.

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

To investigate the role of FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) signaling during pregnancy, mice were injected with anti-FLT1 neutralizing antibody (Ab) beginning on Gestational Day 8 or 12 and every other day thereafter until Day 18; vehicle-only injected mice served as controls. Uterine artery blood flow was measured with ultrasound on Days 13 and 18, and morphometric measurements of the uterine arcade were carried out on Day 19 to provide a measure of gestational vascular remodeling; reproductive performance was evaluated by determining litter size, resorption rates, and pup and placental weights. Ab injections beginning on Day 8 or Day 12 resulted in significant reductions of uterine artery peak systolic and diastolic flows at Days 13 and 18. In addition, normal reproductive function was compromised, as evidenced by a significant reduction in average number of viable pups along with enhanced resorption rates. Reproductive performance was also significantly compromised in this group, although less severely. There was no evidence of a reduction in main uterine artery diameters, though arterial distensibility was reduced, and the diameter of the main uterine vein was significantly smaller in the Ab-injected mice. Significant reductions in main uterine artery and segmental artery length were also noted. Placental and pup weights were similar in all the groups. FLT1 inhibition during murine pregnancy impaired blood flow to the fetal-placental unit, compromised several indices of vascular remodeling, reduced fecundity, and increased fetal reabsorptions. The effects of FLT1 inhibition are most pronounced when targeted during early pregnancy.

Most individuals of the loach Misgurnus anguillicaudatus reproduce bisexually, but cryptic clonal lineages reproduce by natural gynogenesis of unreduced diploid eggs that are genetically identical to maternal somatic cells. Triploid progeny often occur by the accidental incorporation of a sperm nucleus into diploid eggs. Sex reversal from a genetic female to a physiological male is easily induced in this species by androgen treatment and through environmental influences. Here, we produced clonal tetraploid individuals by two methods: 1) fertilization of diploid eggs from a clonal diploid female with diploid sperm of a hormonally sex-reversed clonal diploid male and 2) artificial inhibition of the release of the second polar body in eggs of clonal diploid females just after initiation of gynogenetic development. There is no genetic difference between the clonal diploid and tetraploid individuals except for the number of chromosome sets or genomes. Clonal tetraploid males never produced unreduced tetraploid sperm, only diploid sperm that were genetically identical to those of a clonal diploid. Likewise, clonal tetraploid females did not form unreduced tetraploid eggs, just diploid eggs. However, the eggs' genotypes were identical to those of the original clone, and almost all the eggs initiated natural gynogenesis. Thus, gametogenesis of the clonal tetraploid loach is controlled by the presence of two chromosome sets to pair, thereby preserving the normal meiotic process, i.e., the formation of bivalents and subsequently two successive divisions.

In hermaphroditic fish, the ovotestis can respond to external stimuli so that only one type of gonadal tissue (either ovarian or testicular tissue) will remain reproductively active and the other will recede to a rudimentary stage. However, the molecular mechanism for sexual fate determination is still poorly understood in hermaphroditic fish. In the present study, we examined whether sexual fate determination with respect to testis development is due to differential expression of dmrt1. Expression of dmrt1 was limited to the spermatogonia-surrounding cells (Sertoli cells) throughout testis development. Testicular dmrt1 was differentially expressed in fish (black porgy [Acanthopagrus schlegeli Bleeker]) depending on if fish were destined to be female or male. Expression of dmrt1 in Sertoli cells did not require germ cell factors with busulfan treatment. To examine the role of dmrt1, we used virus-based RNA interference. Deficiency of dmrt1 resulted in a reduced number of germ cells in the testis and stimulated a male-to-female sex change. Higher serum luteinizing hormone levels were detected in 2 - to 3-yr-old male fish as compared to sex-changing female fish. Furthermore, we showed that fish treated in vivo with gonadotropin-releasing hormone (Gnrh) and fish treated in vitro with gonadotropin (Gth) had higher dmrt1 expression in the testis, suggesting that these endocrine factors may affect the male-to-female sex change. Therefore, our data suggest that dmrt1 plays a key role in initial testis differentiation and in later maintenance of male development. We show, to our knowledge for the first time, the functions of dmrt1 in hermaphroditic fish, which indicate that male-phase maintenance may be regulated by the brain-pituitary-gonadal axis via the Gnrh-Gth-Dmrt1 axis.

To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.

The N-ethyl-N-nitrosourea-induced repro42 mutation, identified by a forward genetics strategy, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression.