The pituitary gland is composed of hormone-producing cells essential for homeostasis and reproduction. Pituitary cells are sensitive to endocrine feedback in the adult and can have altered hormonal secretion from exposure to the endocrine disruptor bisphenol A (BPA). BPA is a prevalent plasticizer used in food and beverage containers, leading to widespread human exposure. Although prenatal exposure to BPA can impact reproductive function in the adult, the effects of BPA on the developing pituitary are unknown. We hypothesized that prenatal exposure to low doses of BPA impacts gonadotroph cell number or parameters of hormone synthesis. To test this, pregnant mice were administered 0.5 μg/kg/day of BPA, 50 μg/kg/day of BPA, or vehicle beginning on Embryonic Day 10.5. At parturition, pituitaries from female offspring exposed in utero to either dose of BPA had increased proliferation, as assessed by mKi67 mRNA levels and immunohistochemistry. Coincidently, gonadotroph number also increased in treated females. However, we observed a dichotomy between mRNA levels of Lhb and Fshb. Female mice exposed to 0.5 μg/kg/day BPA had increased mRNA levels of gonadotropins and the gonadotropin-receptor hormone (GNRH) receptor (Gnrhr), which mediates GNRH regulation of gonadotropin production and release. In contrast, mice treated with 50 μg/kg/day of BPA had decreased gonadotropin mRNA levels, Gnrhr and Nr5a1, a transcription factor required for gonadotroph differentiation. No other pituitary hormones were altered on the day of birth in response to in utero BPA exposure, and male pituitaries showed no change in the parameters tested. Collectively, these results show that prenatal exposure to BPA affects pituitary gonadotroph development in females.
Developmental exposure to low doses of bisphenol A increases proliferation and alters gonadotroph differentiation in the pituitary.
anterior pituitary; bisphenol A (BPA); development; developmental biology; endocrine disruptors; environmental contaminants and toxicants; follicle-stimulating hormone (FSH); gonadotrophs; gonadotropins; luteinizing hormone (LH); pituitary
Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.
Aurora-A protein kinase, activated very early in microtubule organizing centers in mouse oocytes, regulates multiple aspects of their biogenesis and spindle formation but does not trigger resumption of meiosis in vivo.
AURKA; CDC25B; centrosome; γ-tubulin; mouse oocytes; MTOC; resumption of meiosis; spindle formation
Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs. The cultured cells grow as clusters of a dynamic mixture of “true” stem cells and differentiating progenitor cells. Cells in the stem/progenitor culture system share many properties with spermatogonia in vivo; however, to fully exploit it as a model for spermatogonial development, new assays are needed that account for the dynamic heterogeneity inherent in the culture system. Here, assays were developed for quantifying dynamics of cultures of stem/progenitor cells that expressed histone-green fluorescent protein (GFP). First, we built on published results showing that cluster formation in vitro reliably predicts the relative number of SSCs. The GFP-based in vitro cluster assay allows quantification of SSCs with significantly fewer resources than a transplantation assay. Second, we compared the dynamics of differentiation in two experimental paradigms by imaging over a 17-day time frame. Finally, we performed short-term live imaging and observed cell migration, coordinated cell proliferation, and cell death resembling that of spermatogonia in the testes. The methods that we present provide a foundation for the use of fluorescent reporters in future microscopy-based high-throughput screens by using living spermatogonial stem/progenitor cultures applicable to toxicology, contraceptive discovery, and identification of regulators of self-renewal and differentiation.
Live imaging of cell proliferation and cell death in spermatogonial stem/progenitor cell cultures elucidates properties of cluster formation.
cell death; differentiation; germ cell; green fluorescent protein; lentivirus; live imaging; spermatogenesis; spermatogonial stem cell; undifferentiated spermatogonia
Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca2+ ([Ca2+]i); however, very few studies exist on [Ca2+]i dynamics in these cells. Other tissues display Ca2+ oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca2+ imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca2+]i changes of living germ cells in situ within the SST preparation. Ca2+ imaging revealed that a subpopulation of male germ cells display spontaneous [Ca2+]i fluctuations resulting from Ca2+ entry possibly throughout CaV3 channels. These [Ca2+]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca2+ fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca2+ oscillations for at least 10 min. Synchronous Ca2+ oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca2+]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca2+ signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.
Male germ cells display coupled spontaneous Ca2+ oscillations recorded in seminiferous tubules slices; these oscillations are due mainly to Ca2+ entry through CaV channels, and gap junctions participate in this coupling.
calcium; cell coupling; intercellular communication; spermatogenesis; testis
Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.
Splice variants of the TREK-1 stretch-activated K+ channel may be associated with preterm labor.
human; labor; myometrial smooth muscle; pregnancy; potassium channels; preterm labor; splice variants; TREK-1; uterus
Repropedia (repropedia.org) is a comprehensive, reproductive-focused online lexicon created to increase public literacy in the fields of reproductive health and science.
Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.
Residues Cys102, Tyr107, and Phe117 within EPPIN's Kunitz domain are critical for binding semenogelin I and could be targets for contraceptive drug design.
contraception; EPPIN; semenogelin I; spermatozoa
Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1–7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.
Establishment of cohesion 1 homolog 2 (ESCO) co-localizes with histone H2AFX is consistent with ESCO2 being present in areas of double-stranded breaks.
germ cells; meiosis; ovary; testis
In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.
The DNA replication licensing protein MCM7 binds to DNA before origin recognition complex, subunit 2 (ORC2), and ORC2 appears to play significant roles in chromatin organization and nuclear structure.
chromatin; DNA replication; early development; fertilization
Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1–100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1–100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96–120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.
Bisphenol A inhibits antral follicle growth by aberrant up-regulation cell cycle regulators, inducing the cell cycle inhibitor transformation-related protein 53 and inducing atresia, independently of the genomic estrogenic pathway.
atresia; bisphenol A; cell cycle; follicle growth
Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based on conserved domains and categorize as the short variant that is devoid of the O-glycan segment. Phylogenetic analysis places LMB Vtgr sequence into a well-supported monophyletic group of fish Vtgr. Real-time PCR showed that the greatest levels of LMB vtgr mRNA expression occurred in previtellogenic ovarian tissues. In addition, we reveal the effects of insulin, 17beta-estradiol (E2), and 11-ketotestosterone (11-KT) in modulation of vtgr, esr, and ar mRNAs in previtellogenic oocytes. Insulin increased vtgr expression levels in follicles ex vivo while exposure to E2 or 11-KT did not result in modulation of expression. However, both steroids were able to repress insulin-induced vtgr transcript levels. Coexposure with insulin and E2 or of insulin and 11-KT increased ovarian esr2b and ar mRNA levels, respectively, which suggest a role for these nuclear receptors in insulin-mediated signaling pathways. These data provide the first evidence for the ordered stage-specific expression of LMB vtgr during the normal reproductive process and the hormonal influence of insulin and sex steroids on controlling vtgr transcript levels in ovarian tissues.
The sex steroids 17beta-estradiol and 11-ketotestosterone repress insulin-induced vitellogenin receptor mRNA expression in previtellogenic oocytes of largemouth bass.
insulin; oocyte development; steroid hormones; vitellogenin; vitellogenin receptor
Accumulating evidence strongly supports the premise that testosterone may be a key player in fetal programming on hypertension. Studies have shown that gestational protein restriction doubles the plasma testosterone levels in pregnant rats. In this study, we hypothesized that elevated testosterone levels in response to gestational protein restriction were caused by enhanced expression of steroidogenic enzymes or impaired expression of Hsd17b2, a known testosterone inactivator that converts testosterone to androstenedione in placenta. Pregnant Sprague-Dawley rats were fed normal (20% protein, control; n = 10) or a low-protein diet (6% protein, PR; n = 10) from Day 1 of pregnancy until killed at Days 14, 18, or 21. Junctional (JZ) and labyrinth (LZ) zones of placenta were collected for expression assay on steroidogenic genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b2, and Srd5a1) by real-time PCR. The main findings include the following: 1) expressions of Cyp11a1, Hsd3b1, and Cyp17a1 in JZ were not affected by diet but were affected by day of pregnancy; 2) expression of Hsd17b2 in both female and male JZs was remarkably increased by PR at Days 18 and 21 of pregnancy; 3) expressions of Hsd17b2 were reduced by PR in both female and male LZ at Day 18 of pregnancy and in female LZ at Day 21 of pregnancy; and 4) expression of Srd5a1in LZ was not affected by day of pregnancy, gender, or diet. These results indicate that in response to gestational protein restriction, Hsd17b2 may be a key regulator of testosterone levels and associated activities in placental zones, apparently in a paradoxical manner.
In the rat, gestational protein restriction reduces expression of Hsd17b2 in the placental labyrinth zone in late pregnancy, allowing testosterone to infuse into fetal circulation and contribute to fetal programming of hypertension.
androgen; gestational protein restriction; Hsd17b2; junctional zone; labyrinth zone; placenta; pregnancy; rat; steroidogenic gene; testosterone
Goodson et al. used a battery of glycolysable and nonglycolysable metabolic substrates to analyze the contribution of glycolysis and oxidative phosphorylation in ATP formation, sperm motility, hyperactivation, and the capacitation-associated increase in protein tyrosine phosphorylation.
The sperm connecting piece is a complex structure that, from a mechanical perspective, appears to play a role in stabilizing the proximal part of the sperm tail. We report the three-dimensional structure of the intact bovine sperm connecting piece, revealing an intricate, asymmetrical architecture with the segmented columns held together by filamentous linkages. The columns fuse, at the proximal end, with each other into structures that form the centriolar vault, and at the distal end, with the outer dense fibers (ODFs). The grouping of the fibers into these structures is consistent with bending only in the plane of the head. Structures reminiscent of the proximal centriole were observed in the vault, while the association of a novel bar structure with ODFs 3 and 8 organizes the distal centriolar vault. It has been proposed that the elastic compliance of the connecting piece provides the underlying mechanism behind initiation of the sperm beat cycle and bend propagation. According to the basal sliding theory of sperm movement, distortion of the connecting piece may store energy that initiates a new beat. The intersegment linkers could serve as mechanosensitive elements that regulate alternation of the sperm tail's bending direction in the beat cycle in addition to providing structural stabilization for the connecting piece segmented structures. On the other hand, our video recordings of the bull sperm movement show little bending of the head with respect to the tail, so it appears that there may be normally little strain within the connecting piece.
Electron cryotomography of the structure of the sperm connecting piece yields novel insights into the motility of spermatozoa.
axoneme; centriole; electron cryotomography; flagella; outer dense fiber; sperm neck
Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.
Both glycolytic and nonglycolytic substrates exhibit differential effects on sperm motility, hyperactivation, and tyrosine phosphorylation during capacitation, and differences between glucose and fructose are correlated with alteration of redox pathways.
glycolysis; metabolism; sperm capacitation; sperm hyperactivation; sperm motility and transport
Hypothalamic neurons, which produce the kisspeptin family of peptide hormones (Kp), are critical for initiating puberty and maintaining estrous cyclicity by stimulating gonadotropin-releasing hormone (GnRH) release. Conversely, RFamide-related peptide-3 (RFRP3) neurons inhibit GnRH activity. It has previously been shown that neonatal exposure to bisphenol A (BPA) can alter the timing of female pubertal onset and induce irregular estrous cycles or premature anestrus. Here we tested the hypothesis that disrupted ontogeny of RFamide signaling pathways may be a mechanism underlying advanced puberty. To test this, we used a transgenic strain of Wistar rats whose GnRH neurons express enhanced green fluorescent protein. Pups were exposed by daily subcutaneous injection to vehicle, 17beta-estradiol (E2), 50 μg/kg BPA, or 50 mg/kg BPA, from Postnatal Day (PND) 0 through PND 3, and then cohorts were euthanized on PNDs 17, 21, 24, 28, and 33 (5–8 animals per age per exposure; males were collected on PNDs 21 and 33). Vaginal opening was advanced by E2 and 50 μg/kg BPA. On PND 28, females exposed to E2 and 50 μg/kg BPA had decreased RFRP-3 fiber density and contacts on GnRH neurons. RFRP3 perikarya were also decreased in females exposed to 50 μg/kg BPA. Data suggest that BPA-induced premature puberty results from decreased inhibition of GnRH neurons.
Premature puberty induced by neonatal bisphenol A exposure results from decreased inhibition of GnRH neurons rather than their increased stimulation.
endocrine disruptor; kisspeptin; puberty; RFRP
Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.
The proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.
caveolae; caveolin-1; FRS2α; placental endothelial cells; proximal FGF2/FGFR1 signaling
Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.
Follistatin overexpression in the testis and epididymis of transgenic mice causes infertility due to defects in fluid resorption, sperm transit, and morphology in excurrent ducts.
epididymis; fertility; follistatin; mouse; testis
During pregnancy, cells from each fetus travel into the maternal circulation and organs, resulting in the development of microchimerism. Identification of the cell types in this microchimeric population would permit better understanding of possible mechanisms by which they affect maternal health. However, comprehensive analysis of fetal cells has been hampered by their rarity. In this study, we sought to overcome this obstacle by combining flow cytometry with multidimensional gene expression microarray analysis of fetal cells isolated from the murine maternal lung during late pregnancy. Fetal cells were collected from the lungs of pregnant female mice. cDNA was amplified and hybridized to gene expression microarrays. The resulting fetal cell core transcriptome was interrogated using multiple methods including Ingenuity Pathway Analysis, the BioGPS gene expression database, principal component analysis, the Eurexpress gene expression atlas, and primary literature. Here we report that small numbers of fetal cells can be flow sorted from the maternal lung, facilitating discovery-driven gene expression analysis. We additionally show that gene expression data can provide functional information about fetal cells. Our results suggest that fetal cells in the murine maternal lung are a mixed population, consisting of trophoblasts, mesenchymal stem cells, and cells of the immune system. Detection of trophoblasts and immune cells in the maternal lung may facilitate future mechanistic studies related to the development of immune tolerance and pregnancy-related complications, such as pre-eclampsia. Furthermore, the presence and persistence of mesenchymal stem cells in maternal organs may have implications for long-term postpartum maternal health.
Comprehensive gene expression microarray analysis of fetal cells isolated from the pregnant murine maternal lung shows the major cell populations to be trophoblasts, mesenchymal stem cells, and cells of the immune system.
mesenchymal stem cells; microarray; microchimerism; reproductive immunology; trophoblasts
SLC2A8, also known as GLUT8, is a facilitative glucose transporter expressed in the testis, brain, liver, heart, uterus, ovary, and fat. In this study we examined the effect of Slc2a8 deficiency on mouse gamete, preimplantation embryo, and implantation phenotype, as well as postnatal growth and physiology. For this model, the transcriptional start site and exons 1–4 were targeted and a lack of protein expression was confirmed by Western immunoblot. Oocytes obtained from Slc2a8−/− mice demonstrated abnormal metabolism and ATP production. In addition, deletion of Slc2a8 resulted in impaired decidualization, a critical step in the differentiation of endometrial stromal cells (ESCs), necessary for implantation. This indicates a role for SLC2A8 in decidualization, which is supported by Slc2a8 mRNA expression in both mouse and human ESCs, which increases dramatically in response to hormonal changes occurring during the process of implantation. Ovarian transplantation studies confirm that lack of SLC2A8 affects both the embryo and the implantation processes. This phenotype leads to decreased litter size, and smaller pups at weaning that continue to display an abnormally small growth phenotype into adulthood. The Slc2a8 null mice display decreased body fat by magnetic resonance imaging, and, interestingly, they are resistant to a diet high in fat and carbohydrates.
Global knockout of the glucose transporter Slc2a8 leads to growth retardation and small litter size in mice.
glucose homeostasis; glucose transport; GLUT8; growth; SLC2A8; uterine decidualization
The mechanism(s) by which vitamin D3 regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D3 deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D3) null mice to study the effect of vitamin D3 deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D3-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D3 deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D3-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D3-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D3-replete diet. Estrous cycles were restored when vitamin D3-deficient Cyp27b1 null young adult females were transferred to a vitamin D3-replete diet. This study is the first to demonstrate that peripubertal vitamin D3 sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D3 is a key regulator of neuroendocrine and ovarian physiology.
Studies in Cyp27b1 null and wild-type mice show that peripubertal vitamin D3 deficiency delays puberty and disrupts ovarian physiology and estrous cycles.
hypothalamus; nutrition; ovulation; puberty; vitamin D3
Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.
PMID: 22539679 CAMSID: cams3210
IFNγ; implantation failure in type 1 diabetes; MUC1; NFκB; NOD mice; PIASy; progesterone receptor; STAT3; uterodomes
Pre-conception or gestationally-induced diabetes increases morbidities and elevates long-term cardiovascular disease risks in women and their children. Spontaneously hyperglycemic (d)-NOD/ShiLtJ females, a type 1 diabetes model, develop bradycardia and hypotension after midpregnancy compared with normoglycemic, age and gestation day (gd)-matched controls (c-NOD). We hypothesized that onset of the placental circulation at gd9–10 and rapid fetal growth from gd14 correlate with aberrant hemodynamic outcomes in d-NOD females. To develop further gestational time course correlations between maternal cardiac and renal parameters, high-frequency ultrasonography was applied to virgin and gd8–16 d- and c-NODs. Cardiac output and left ventricular (LV) mass increased in c- but not d-NODs. Ultrasound and postmortem histopathology showed overall greater LV dilation in d- than c-NOD mice in mid-late gestation. These changes suggest blunted remodeling and altered functional adaptation of d-NOD hearts. Umbilical cord ultrasounds revealed lower fetal heart rates from gd12 and lower umbilical flow velocities at gd14 and 16 in d- versus c-NOD pregnancies. From gd14–16, d-NOD fetal losses exceeded those of c-NOD. Similar aberrant responses in human diabetic pregnancies may elevate postpartum maternal and child cardiovascular risk, particularly if mothers lack adequate prenatal care or have poor glycemic control over gestation.
PMID: 23636813 CAMSID: cams2944
Pregnancy; diabetes; NOD mouse; cardiovascular system; cardiac adaptations
Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation—geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)—the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.
Simvastatin decreases invasiveness of human endometrial stromal cells and decreases expression of matrix metalloproteinases 2 and 3 and CD44, while increasing expression of tissue inhibitor of matrix metalloproteinase 2.
CD44; endometriosis; human endometrial stromal cells; invasion assay; metalloproteinases; simvastatin; tissue inhibitor of metalloproteinases
Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.
Embryo biopsy deregulates steroid metabolism during mouse pregnancy and leads to decreased fetal body weight.
assisted reproduction technologies; embryo; in vitro fertilization; placenta; steroid hormones