Hypothalamic-hypophysiotropic peptides are the proximate regulators of pituitary cells, but they cannot fully account for the complex functioning of these cells. Accordingly, awareness is growing that an array of peptides produced in the pituitary exert paracrine/autocrine functions. One such peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was originally identified as a hypothalamic activator of cAMP production in pituitary cells. Gonadotrophs and folliculostellate cells are the main source of pituitary PACAP, and each pituitary cell type expresses a PACAP receptor. PACAP increases alpha-subunit (Cga) and Lhb mRNAs, and it stimulates the transcription of follistatin (Fst) that, in turn, restrains activin signaling to repress Fshb and gonadotropin-releasing hormone-receptor (Gnrhr) expression as well as other activin-responsive genes. The PACAP (Adcyap1) promoter is activated by cAMP, and pituitary cells may communicate by a feed-forward, cAMP-dependent mechanism to maintain a high level of PACAP in the fetal pituitary. At birth, pituitary PACAP declines and pituitary follistatin levels decrease, which together with increased gonadotropin-releasing hormone secretion allow Gnrhr and Fshb to increase and facilitate activation of the newborn gonads. Changes in Adcyap1 expression levels in the adult pituitary may contribute to the selective rise in follicle-stimulating hormone (FSH) from age 20–30 days to the midcycle surge and to the secondary increase in FSH that occurs before estrus. These results provide further support for the notion that PACAP is a key player in reproduction through its actions as a pituitary autocrine/paracrine hormone.

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P₄) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P₄ levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.

The Nos3-knockout mouse, deficient for endothelial constitutive nitric oxide synthase (NOS3), is affected by a reduction in the number and weight of the embryos and constitutes a good model for some features of preeclampsia and intrauterine growth retardation (IUGR). Deficiencies in conceptus growth and survival may result from factors inherent in the embryo itself or from deficiencies in uterine function. In the current study, we aimed to determine the effects of embryonic genotype independently of maternal genotype, which can affect uterine environment. Therefore, by using magnetic resonance imaging (MRI), we characterized the phenotypes of NOS3-defective (Nos3^−/−; n = 6), normal wild-type (Nos3 ^/ ; n = 5), and heterozygous (Nos3 ^/−; n = 16) mouse fetuses. All of them were littermates obtained by breeding heterozygous mice (Nos3 ^/−); therefore, the maternal genotype was the same for all the fetuses. At Day 13.5 (i.e., Theiler stage TS 21–22), females were anesthetized and scanned with three-dimensional MRI. Analysis of the different measurements of the embryos and the gestational annexes showed no significant differences between Nos3 ^/ and Nos3 ^/−; however, there was a trend toward larger sizes in Nos3 ^/ , and values in Nos3^−/− were significantly smaller than in Nos3 ^/ and Nos3 ^/−. The reduction in the crown-rump length of Nos3^−/− reached 12% when compared to Nos3 ^/ (P < 0.05); the effect was higher for head measurements (16% for occipito-snout length and biparietal diameter, P < 0.05 for both) and trunk diameter (17%, P < 0.05). Overall, the maximum area of fetuses in longitudinal planes decreased 27% (P < 0.05) when comparing Nos3^−/− to wild-type Nos3 ^/ . Finally, Nos3^−/− showed a reduction of 29% in the maximum thickness of the placenta, which may be related to the appearance of IUGR due to compromised nutritional delivery to the fetus.

Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed using immunohistochemistry and Western blotting. Gene ontology classification of differentially regulated transcripts underlines the ongoing embryo-maternal dialogue. Transcript showing higher expression levels as conceptus' development proceeds mainly localizes to the extracellular environment, thereby having the potential to act upon the uterine environment. Genes involved in the positive regulation of the immune system are enriched among transcripts displaying decreased expression, reflecting the need of the semiallograft conceptus to be protected from the immune system. A subset of differentially expressed genes, such as BRCA1 and FGF2, has previously been described to be expressed by early stages of embryonic development, whereas other transcripts are apparently unique to equine conceptuses, as their expression has not been reported in other species. These transcripts include fibrinogen subunits, the expressions of which were confirmed at the mRNA and protein level. Furthermore, results indicate the counteraction of trophoblast invasion, and that the conceptus appears to regulate changes in sialic acid content of its capsule, an event suggested to be essential for successful establishment of pregnancy.

Retinoic acid (RA) is required for germ cell differentiation, the regulation of which gives rise to a constant production of mature sperm. In testes from 3-day postpartum (dpp) RARE-hsplacZ mice, periodic regions positive for beta-galactosidase activity were observed along the length of the seminiferous tubules. Periodicity was abolished by treatment of neonates with exogenous RA at 2 dpp. To assess the consequences, 2-dpp mice were treated with RA, and the long- and short-term effects were assessed. Long-term effects of neonatal RA exposure included a delay in the appearance of advanced germ cells and the absence of a spermatogenic wave (synchronous spermatogenesis) in the adult. In contrast, RA exposure in vitamin A-sufficient adults did not result in synchronous spermatogenesis but rather induced apoptosis in a subset of spermatogonia. Shortly after (24 h) neonates were exposed, altered expression of known germ cell differentiation and the (Stra8, Kit, Sycp3, and Rec8) meiosis markers and an increase in the number of STRA8 and SYCP3 immunopositive cells were observed relative to those of vehicle controls. However, 48 and 72 h after exposure, a significant reduction in the number of STRA8 and SYCP3 immunopositive cells occurred. Immunohistochemical analysis of a marker for apoptosis demonstrated neonatal exposure resulted in increased germ cell apoptosis, as observed in the adult. Additionally, RA exposure resulted in increased Cyp26a1 expression of the RA-degrading enzyme. Thus, while RA treatment of neonatal and adult mice resulted in apoptosis of spermatogonia, synchronous spermatogenesis occurred only after neonatal RA exposure.

Appearance of mouse fetal Leydig cells requires activation of the Hedgehog pathway. Upon binding to the membrane-bound receptor patched, Hedgehog ligands induce intracellular responses via a combined effect of Gli transcription factors. Szczepny et al. (Biol Reprod 2009; 80:258–263) found that Gli1, one of the three Gli transcription factors, is present in the fetal testis and that its expression is suppressed by the Hedgehog inhibitor cyclopamine. In this study, we investigated the involvement of the Gli1 and Gli2 factors in mouse fetal Leydig cell differentiation. The Gli1 and Gli2 transcription factors showed an overlapping expression pattern in the testis interstitium at the time when fetal Leydig cells appear. Despite their similar expression, Gli1 and Gli2 patterns were differentially regulated. Initial Gli1 and Gli2 expression depends upon an active Hedgehog pathway; however, maintenance of only Gli1, but not Gli2, expression requires activation of the pathway. Inactivation of either the Gli1 or Gli2 gene did not affect fetal Leydig cell development and testis morphology, suggesting a functional redundancy. When the transcriptional activity of both GLI1 and GLI2 was suppressed by a selective inhibitor, GANT61, in cultured fetal testes before the appearance of fetal Leydig cells, Gli1 and Gli2 expression and steroidogenic marker activity were completely abolished. However at later stages when Leydig cells were already present, GANT61 treatment inhibited Gli1 expression but had no effects on Gli2 expression and fetal Leydig cell appearance. Our results reveal overlapping and redundant Gli1 and Gli2 roles in fetal Leydig cell differentiation and a novel regulation of Gli2 expression in the fetal testis.

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.

Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence.

Temporality among episodes of a prostaglandin F2alpha metabolite (PGFM), progesterone (P4), luteinizing hormone (LH), and estradiol (E2) were studied during preluteolysis and luteolysis. A vehicle group (n = 10) and a group with an E2-induced PGFM pulse (n = 10) were used. Blood sampling was done every 0.25 h for 8 h. An episode was identified by comparing its coefficient of variation (CV) with the intra-assay CV. Pulsatility of PGFM, P4, LH, and E2 in individual heifers was inferred if the autocorrelation functions were different (P < 0.05) from zero. About four nonrhythmic fluctuations of PGFM/8 h were superimposed on PGFM pulses. Pulsatility was detected for LH but not for P4 and E2. A transient increase in P4 was not detected during the ascending portion of a PGFM pulse. Progesterone decreased (P < 0.003) during Hours −1.25 to −0.50 of the PGFM pulse (Hour 0 = peak) and ceased to decrease temporally with an increase (P < 0.05) in LH. Maximum P4 concentration occurred 0.25 h after an LH pulse peak, and an increase (P < 0.005) in E2 began at the LH peak. Nadirs of LH pulses were greater (P < 0.05) and the nadir-to-nadir interval was shorter (P < 0.003) in the E2 group, which is consistent with reported characteristics during luteolysis. The results did not support the hypothesis of a transient P4 increase early in a PGFM pulse and indicated a balance between a luteolytic effect of PGF and a luteotropic effect of LH within the hours of a PGFM pulse.

Interferon-tau (IFNT) is the trophoblast-secreted factor responsible for establishing and maintaining pregnancy in ruminants. Several uterine- and embryo-derived factors, including fibroblast growth factor-2 (FGF2), regulate IFNT production. The objective of the present study was to decipher the intracellular signaling mechanisms employed by FGF2 to regulate IFNT production. In bovine trophoblast cells (CT1), mitogen-activated protein kinase kinase-dependent pathways mediated constitutive IFNT mRNA concentrations. However, FGF2-mediated increases in IFNT mRNA levels occurs independent of mitogen-activated protein kinase. Exposure to the pan-protein kinase C (PKC) inhibitor, calphostin C, did not affect basal IFNT mRNA levels but limited the ability of FGF2 to increase IFNT mRNA abundance. Also, supplementation with phorbol 12-myristate 13-acetate (PMA) stimulated IFNT mRNA levels to the same extent as with FGF2. PMA and FGF2 cosupplementation did not elicit an additive effect on IFNT mRNA abundance. Pharmacological antagonists for classic PKCs (Gö6976) or novel PKCs, including PKC delta (rottlerin), were used to identify the specific PKC isoform utilized by FGF2. Supplementation of CT1 cells with Gö6976 did not affect FGF2 or PMA activities, whereas rottlerin prevented FGF2- and PMA-dependent increases in IFNT mRNA abundance in CT1 cells. Rottlerin also prevented FGF2 from increasing IFNT mRNA levels in Vivot trophoblast cells and primary trophoblast outgrowths. Modifications in PRKCD phosphorylation status were evident following FGF2 and PMA treatment. Also, reducing PRKCD expression by RNA interference attenuated FGF2-dependent increases in IFNT mRNA abundance. In conclusion, these results provide evidence that FGF2 regulates IFNT production in bovine trophectoderm by acting through PRKCD.

Ribosomal DNA (rDNA) is not composed of multiple copies of identical transcription units, as commonly believed, but rather of at least seven rDNA variant subtypes that are expressed in somatic cells. This finding raises the possibility that ribosome function may be modulated as proposed by the ribosome filter hypothesis. We report here that mouse oocytes and preimplantation embryos express all the rDNA variants except variant V and that there is no marked developmental change in the qualitative pattern of variant expression. The maternal and embryonic ribosome pools are therefore quite similar, minimizing the likelihood that developmental changes in composition of the ribosome population are critical for preimplantation development.

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.

Nitric oxide (NO) plays a key role in placental biology, and placental dysfunction is the main pathogenesis pathway for preeclampsia, yet the direct placental targets of NO actions have not been determined. Covalent adduction of an NO moiety to cysteines, termed S-nitrosylation (SNO), is emerging as a key route by which NO can directly modulate protein functions. This study was conducted to analyze global S-nitroso (SNO)-proteins in human placentas and to determine if their levels differ in normotensive versus severe preeclamptic placentas. Although total nitrite/nitrate increased, total levels of SNO-proteins and nitrosylated forms of endothelial NO synthase and heat shock protein 90 were decreased by preeclampsia. We further compared normotensive and preeclamptic placental nitroso-proteomes (total SNO-protein profiles) by using a biotin and CyDye switch test combined with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and identified SNO-proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Numerous SNO-proteins were displayed as spots on 2D-DIGE gels. One hundred spots of interest were excised; 46 spots were identified, of which 8 spots were novel SNO-proteins; levels of 15 spots were increased, and 6 spots were decreased, and the rest were unchanged by preeclampsia. Pathway analysis suggested that placental SNO-proteins are involved in regulating various cellular functions including protein synthesis, cell movement and metabolism, cell signaling, and other functions. These data therefore show for the first time that SNO is a crucial mechanism by which NO directly regulates placental proteins linked to various biological pathways. The significantly altered placental nitroso-proteome in preeclampsia suggests that SNO plays a role in the placental pathophysiology in preeclampsia.

Previous studies in MA-10 tumor Leydig cells demonstrated that disruption of the mitochondrial electron-transport chain (ETC), membrane potential (ΔΨ_m), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ΔΨ_m, and ATP synthesis cooperatively affected steroidogenesis. These results suggest significant differences between the two systems and call into question the extent to which results from tumor Leydig cells relate to primary cells. Thus, to further understand the similarities and differences between the two systems as well as the impact of ATP disruption on steroidogenesis, we performed comparative studies of MA-10 and primary Leydig cells under similar conditions of mitochondrial disruption. We show that mitochondrial ATP synthesis is critical for steroidogenesis in both primary and tumor Leydig cells. However, in striking contrast to primary cells, perturbation of ΔΨ_m in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ΔΨ_m powers the mitochondrial ATP synthase. Further studies revealed that a significant proportion of cellular ATP in MA-10 cells derives from glycolysis. In contrast, primary cells appear to be almost completely dependent on mitochondrial respiration for their energy provision. Inhibitor studies also suggested that the MA-10 ETC is impaired. This work underscores the importance of mitochondrial ATP for hormone-stimulated steroid production in both MA-10 and primary Leydig cells while indicating that caution must be exercised in extrapolating data from tumor cells to primary tissue.

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated. In the present study, we examined the roles of THY1 and integrin beta3 in theca cell layer formation during mouse folliculogenesis. The localization pattern of THY1 and integrin beta3 in adult mouse ovary was investigated immunohistochemically. The strongest THY1 signal was observed in theca cell layers from secondary to preantral follicles, at which time theca cells have begun to participate in follicle formation. Integrin beta3 also localized to the theca cell layer of secondary to preantral follicles and showed a localization pattern similar to that of THY1. Moreover, the role of THY1 in theca cell layer formation was examined using a follicle culture system. When anti-THY1 antibody was added to this culture, no theca cell layers were formed, and the granulosa cells were distanced from each other. Because a THY1 signal was not observed in ovaries at stages earlier than prepuberty, THY1 localization also appeared to be affected by mouse development. This possibility was examined by determining the effect of administering follicle-stimulating hormone, luteinizing hormone, and 17beta-estradiol to 7-day-old mice on THY1 localization in the ovary 3 days later. Only follicle-stimulating hormone induced a THY1 signal in 10-day-old mouse ovaries. No THY1 signal was observed in untreated 10-day-old ovaries. In conclusion, THY1 might play a role in cell adhesion via binding to integrin beta3 in mouse ovaries. The present results suggest that THY1 localization may be affected by follicle-stimulating hormone in mouse ovaries.

In pituitary gonadotrophs, GnRH induces expression of the mitogen-activated protein kinases (MAPK3/1) dephosphorylating enzyme, dual-specificity phosphatase 1 (DUSP1). Here we examined DUSP1 expression levels following pulsatile GnRH stimulation of the LbetaT2 gonadotroph cells. DUSP1 expression was increased more prominently following high-frequency (every 30 min) GnRH pulse stimulation (7.02- ± 1.47-fold) than low-frequency (every 120 min) GnRH pulses (2.68- ± 0.09-fold). With high-frequency GnRH pulses, DUSP1 expression increased by 2.89- ± 0.32-fold 2 h after GnRH pulse initiation (four 5-min pulses). DUSP1 expression was not induced following lower frequency GnRH pulses, even when the GnRH concentration was increased. Under high-frequency conditions, MAPK3/1 phosphorylation was observed 10 min after the GnRH pulse and decreased to basal levels after 25 min. However, MAPK3/1 dephosphorylation did not occur concurrently with DUSP1 expression. Overexpression of MAP3K1, a kinase upstream of MAPK3/1, increased both the Lhb and the Fshb subunit promoter activities, which could be completely inhibited by cotransfection with DUSP1-expressing vectors. Serum response factor (Srf) promoter activities induced by MAP3K1 were also prevented by DUSP1 overexpression, confirming that MAPK3/1 has an important role in gonadotropin subunit gene expression. Both high- and low-frequency GnRH pulse stimulation failed to increase the Lhb and Fshb subunit gonadotropin gene expression levels upon DUSP1 overexpression. Our study demonstrates that DUSP1 is specifically expressed following high-frequency GnRH pulses and that this effect may participate in the differential regulation of gonadotropin subunit expression in association with MAPK3/1 phosphorylation.

Motile cilia and flagella exhibit many waveforms as outputs of dynein activation sequences on the highly conserved axoneme. Motility change of sperm in the reproductive tract is difficult to study and remains an important area of investigation. Sperm typically execute a sinusoidal waveform. Increased viscosity in the medium induces somewhat unusual arc-line and helical waveforms in some sperm. However, whether the latter two waveforms occur in vivo is not known. Using green fluorescence protein imaging, we show that Drosophila sperm in the uterus move in circular foci via arc-line waves, predominantly in a tail-leading orientation. From the uterus, a small fraction of the sperm enters the seminal receptacle (SR) in parallel formations. After sperm storage and coincident with fertilization of the egg, the sperm exit the SR via head-leading helical waves. Consistent with the observed bidirectional movements, the sperm show the ability to propagate both base-to-tip and tip-to-base flagellar waves. Numerous studies have shown that sperm motility is regulated by intraflagellar calcium concentrations; in particular, the Pkd2 calcium channel has been shown to affect sperm storage. Our analyses here suggest that Pkd2 is required for the sperm to adopt the correct waveform and movement orientation during SR entry. A working model for the sperm's SR entry movement is proposed.

Perfluorooctanesulfonate (PFOS) was produced and used by various industries and in consumer products. Because of its persistence, it is ubiquitous in air, water, soil, wildlife, and humans. Although the adverse effects of PFOS on male fertility have been reported, the underlying mechanisms have not yet been elucidated. Here, for the first time, the effects of PFOS on testicular signaling, such as gonadotropin, growth hormone, insulin-like growth factor, and inhibins/activins were shown to be directly related to male subfertility. Sexually mature 8-wk-old CD1 male mice were administered by gavages in corn oil daily with 0, 1, 5, or 10 mg/kg PFOS for 7, 14, or 21 days. Serum concentrations of testosterone and epididymal sperm counts were significantly lower in the mice after 21 days of the exposure to the highest dose compared with the controls. The expression levels of testicular receptors for gonadotropin, growth hormone, and insulin-like growth factor 1 were considerably reduced on Day 21 in mice exposed daily to 10 or 5 mg/kg PFOS. The transcript levels of the subunits of the testicular factors (i.e., inhibins and activins), Inha, Inhba, and Inhbb, were significantly lower on Day 21 of daily exposure to 10, 5, or 1 mg/kg PFOS. The mRNA expression levels of steroidogenic enzymes (i.e., StAR, CYP11A1, CYP17A1, 3beta-HSD, and 17beta-HSD) were notably reduced. Therefore, PFOS-elicited subfertility in male mice is manifested as progressive deterioration of testicular signaling.

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.

Transcription factor GATA4 is expressed in granulosa cells and, to a lesser extent, in other ovarian cell types. Studies of mutant mice have shown that interactions between GATA4 and its cofactor, ZFPM2 (also termed FOG2), are required for proper development of the fetal ovary. The role of GATA4 in postnatal ovarian function, however, has remained unclear, in part because of prenatal lethality of homozygous mutations in the Gata4 gene in mice. To circumvent this limitation, we studied ovarian function in two genetically engineered mouse lines: C57BL/6 (B6) female mice heterozygous for a Gata4-null allele, and 129;B6 female mice in which Gata4 is deleted specifically in proliferating granulosa cells using the Cre-loxP recombination system and Amhr2-cre. Female B6 Gata4 ^/− mice had delayed puberty but normal estrous cycle lengths and litter size. Compared to wild-type mice, the ovaries of gonadotropin-stimulated B6 Gata4 ^/− mice were significantly smaller, released fewer oocytes, produced less estrogen, and expressed less mRNA for the putative GATA4 target genes Star, Cyp11a1, and Cyp19. Gata4 conditional knockout (cKO) mice had a more severe phenotype, including impaired fertility and cystic ovarian changes. Like Gata4 ^/− mice, the ovaries of gonadotropin-stimulated cKO mice released fewer oocytes and expressed less Cyp19 than those of control mice. Our findings, coupled with those of other investigators, support the premise that GATA4 is a key transcriptional regulator of ovarian somatic cell function in both fetal and adult mice.

Resveratrol (trans-3,4N-trihydroxystilbene), a phytoalexin present in grapes and red wine is emerging as a natural compound with anticancer properties. However, the physiological and molecular effects of resveratrol on normal uterine cells are poorly understood. In the present study we evaluated the effects of resveratrol on normal uterine cells and the mechanisms involved in vivo. Healthy immature rats were treated s.c. with resveratrol (0, 0.5, 5, and 50 mg/kg body weight) for 7 consecutive days and euthanized on the eighth day. Uteri were collected and weighed, and endometrium was recovered for total protein extraction, followed by Western blot analysis. Estrogen receptor alpha 1 (ESR1) and beta 2 (ESR2) affinity and activation by resveratrol were also determined by in vitro ESR-binding assays. Immunohistochemistry (IHC) studies were performed to visualize the proliferation marker, proliferating cell nuclear antigen (PCNA), and immunofluorescence (IF) studies were done to study the localization of PTGS2. The results showed that resveratrol increased uterine wet weight and uterine body weight ratios significantly. This local cellular proliferation in terms of the thickening of the columnar epithelial cells and an increase in the number of glands was accompanied by an increase of AKT 16 phosphorylation and PTGS2 and XIAP protein expression. These results were further supported by IF and IHC analyses. Total AKT, ESR1, and ESR2 protein expression levels were not modulated by the treatment; however, resveratrol showed moderate estrogenicity for both ESR isoforms. Expression of progesterone receptor A (PGR) was induced in the presence of resveratrol. These data support the hypothesis that resveratrol can act in a prosurvival or antiapoptotic way through AKT, XIAP, and PTGS2 regulation in the endometrium and could positively affect the outcome of pregnancy and favor fertility.

A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C.

Though the decidua serves a critical function in implantation, the hormonal regulated pathway in decidualization is still elusive. Here we describe in detail the regional distribution and the effects of progesterone receptors (PGR), estrogen receptors (ESR), and MAPK activation on decidualization. We showed an increase in PGR A, PGR B, ESR1, and phosphorylated MAPK3–1 proteins (p-MAPK3–1), but not in ESR2, in the decidual tissue up to Day 8 of pregnancy. PGR was predominantly found in the nuclei of mesometrial decidual cells and of undifferentiated stromal cells where it colocalizes with ESR2 and ESR1. In the antimesometrial decidua, all the receptors showed cytoplasmic localization. MAPK was activated exclusively in undifferentiated stromal cells of the junctional zone between the antimesometrial and mesometrial decidua and at the border of the antimesometrial decidua. Treatment with the progesterone antagonist onapristone and/or the estrogen antagonist faslodex reduced the extent of decidual tissue and downregulated the levels of PGR and ESR1. The expression level of ESR2 was affected only by the progesterone receptor antagonist, while neither the antiprogestin nor the antiestrogen significantly modified the p-MAPK3–1 level. The inhibition of MAPK3–1 phosphorylation by PD98059 impaired the extent of decidualization and the closure reaction of the implantation chamber, and significantly downregulated ESR1. These results confirm a role of both steroid receptors in the growth and differentiation of the different decidual regions and suggest a new function for p-MAPK3–1 in regulating expression levels of ESR1, thereby maintaining the proliferation capacity of stromal cells and limiting the differentiation process in specified regions of decidual tissues.