Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshall's Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1–126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.

Gonadotropin-releasing hormone (GNRH) is a neuropeptide critical for reproductive activation and maintenance in vertebrates. The recent elucidation of molluscan GNRH-like sequences led to several important questions regarding the evolution of the GNRH family. For instance, are molluscan and chordate GNRHs true orthologs? Has GNRH been retained in most protostomian lineages? What was the function of the ancestral GNRH? The goal of this review is to provide a critical analysis of GNRH evolution based on data available from the known forms of protostomian GNRH. Judging from the orthology between chordate and protostomian GNRH receptors, conservation of several structural motifs on the GNRH peptide, and exon/intron arrangement conserved between protostomian and chordate GNRH genomic sequences, we conclude that chordate and protostomian GNRHs likely share a common ancestor. Based on our analysis of phylogenetic distribution, we also hypothesize that GNRH may have been lost in the ecdysozoan lineage but preserved in lophotrochozoans. Lastly, we propose that the ancestral function of GNRH is to serve as a general neural regulator, and its considerable specialization in reproduction seen in chordates is a consequence of neofunctionalization following gene duplication..

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T_e) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H_e) alone group and by Day 8 in the T_e alone group but peaked at Day 3 in H_e T_e group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P < 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling..

Fusion of cytotrophoblasts into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. The envelope protein of a human endogenous retrovirus W (HERV-W) family member, syncytin 1, has been shown to mediate placental cell fusion. Recently, the envelope protein of another HERV family member (HERV-FRD), syncytin 2, has been identified and shown to be highly expressed in the placenta. To better understand the biology of syncytin 2, in this study we first investigated syncytin 2 gene expression in normal and preeclamptic placentas and then characterized the functions of syncytin 2. The expression of syncytin 2 gene was decreased in preeclamptic placentas and could be stimulated by the cAMP stimulant forskolin. The endoprotease furin was found to be involved in the posttranslational cleavage of syncytin 1 and 2 polypeptides into surface and transmembrane subunits. In addition, proper association of the subunits of syncytins 1 and 2 is probably required for the functional integrity of each protein, because subunit swapping of syncytins 1 and 2 failed to generate fusogenic chimeras. Finally, we demonstrated that the disulfide bridge-forming CX₂C and CX₇C motifs found in syncytins 1 and 2 are essential for their fusogenic activities, because mutations in the CX₂C motif not only abolished fusogenesis but also functioned as dominant-negative mutants. Our results suggest that syncytin 2 may function as a second fusogenic protein for placental cell fusion..

Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency..

Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods..

The purpose of the study was to understand in more detail the natural history of fetomaternal cell trafficking in healthy pregnant mice. Our goal was to identify the best target organs and days during pregnancy for further mechanistic studies of the role of fetal cells in maternal disease and injury. C57BL/6J wild-type virgin females (n = 54) were mated with congenic enhanced green fluorescent protein (EGFP) transgenic males. During pregnancy and after delivery, female mice were euthanized, and eight organs and blood were analyzed for the presence of fetal GFP cells with flow cytometry and real-time quantitative PCR. Maternal lungs, liver, and spleen were also analyzed by fluorescent stereomicroscopy. Fetal GFP cells were first found at low frequency at Embryonic Day 11, increased to a maximum at Embryonic Day 19, and decreased rapidly postpartum. These fetal cell dynamics were significantly reproducible among all mice studied. In addition, there was a consistent distribution of fetal cells within maternal organs, with lung, liver, blood, and spleen having the greatest concentrations; these were highly correlated at all time points (P < 0.0001). Maternal lung contained 10- to 100-fold more fetal cells than any other organ, and using all three techniques, the number of fetal cells detected was the most consistent and reproducible in this organ. Stereomicroscopy showed that within the lung, fetal cells were widely and apparently randomly distributed. Using a murine model, our data demonstrate that fetomaternal cellular trafficking occurs in reproducible patterns, is maximal near term delivery, and has predilection for the maternal lung..

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4 and CD8 T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure..

Male peacock blennies (Salaria pavo) release odors from their anal glands and, possibly, from their gonads that attract reproductive females. The current study investigated the effects of 11-ketotestosterone (KT) on development of the anal glands, testes, and accessory testicular organs as well as the subsequent olfactory potency of their secretions. After 3 wk of KT treatment (5 mg/kg in silastic implants), clusters of cells secreting neutral mucins differentiated in the anal gland of all treated males, whereas this occurred in only one control male. Secretions by anal glands from KT-treated males elicited greater olfactory responses, as assessed by electro-olfactography, than those from controls. Treatment with KT stimulated testicular gland growth and sialomucin secretion but had no clear effect on the germinal region of the testis; KT also stimulated enlargement of, and fluid secretion in, the blind pouches (paired evaginations of the spermatic ducts). Secretions by the testes and fluid in blind pouches from KT-treated males elicited greater olfactory responses than those from controls. In conclusion, KT stimulates development of the anal glands and testicular accessory organs and promotes odorant secretion, the putative multicomponent male peacock blenny pheromone..

Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca² channels, and extracellular Ca² dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca² channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding..

Hypothalamic glutamate and gamma-aminobutyric acid (GABA) neurotransmission are involved in the ovarian hormone-induced GnRH-LH surge in rodents. We previously reported that middle-aged rats have significantly less glutamate release in the medial preoptic area than young rats on the day of the LH surge. The present study tested the hypothesis that the delayed and attenuated LH surge in ovariohysterectomized middle-aged rats primed with ovarian steroids results from reduced hypothalamic glutamate and increased GABA_A neurotransmission. Microdialysis results show that middle-aged rats with attenuated LH surges had reduced extracellular glutamate and increased extracellular GABA levels in the medial preoptic area compared with young rats. Blocking GABA_A receptors with bicuculline or inhibiting synaptic glutamate reuptake with l-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular Glu in the medial preoptic area and partially restored LH surge amplitude in middle-aged rats without altering LH surge onset. Complete recovery of LH surge amplitude was observed in middle-aged rats treated with the combination of bicuculline and l-trans-pyrrolidine-2,4-dicarboxylic acid. This treatment also restored the extracellular glutamate:GABA ratio in the medial preoptic area of middle-aged rats to the level of young rats. Immunoblot analysis revealed that estradiol and progesterone treatment reduced SLC32A1(formerly known as vesicular GABA transporter) levels and increased SLC17A6 (formerly known as vesicular glutamate transporter 2) levels in the anterior hypothalamus of ovariohysterectomized young but not middle-aged rats. These data suggest that both reduced availability of glutamate and increased activation of GABA_A receptors under estrogen-positive feedback conditions contribute to the age-related delay in onset and attenuated amplitude of the LH surge..

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific..

The role of androgen and androgen receptors (ARs) in males has been well established. This steroid and its receptor also exist in follicles, but their functions are still unclear. In this study, using a culture system containing a low dose of hypoxanthine, we revealed the positive contribution of testosterone to oocyte meiotic resumption. By performing ultracentrifugation to allow clear visualization of porcine germinal vesicles, our results provide evidence that mitogen-activated protein kinase (MAPK) in the oocyte itself but not in cumulus cells was activated before germinal vesicle breakdown (GVBD) after testosterone treatment. We further explored the signal cascade of testosterone-triggered GVBD and showed significant contributions of AR to testosterone-induced MAPK activation and GVBD. By using a potent and selective inhibitor of SRC and detecting activation of the kinase, we found that testosterone activated SRC in oocytes but not in cumulus cells and that SRC (as an essential upstream molecule of MAPK) mediated this testosterone- and AR-promoted reinitiation of meiosis. The present findings propose an undefined signaling pathway and suggest the potential competence of testosterone for meiotic resumption in mammalian oocytes..

Sumoylation is a posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are covalently attached to their substrates. In vertebrates, developmental roles for sumoylation have been studied, but the function of sumoylation during mammalian oocyte growth and maturation is not known. As a prelude to conducting studies on the role of sumoylation during oocyte development, we analyzed the temporal and spatial pattern of expression of UBE2I, a SUMO-conjugating E2 enzyme. Immunocytochemical analysis of UBE2I revealed a punctate nuclear staining pattern, with transcriptionally quiescent, fully grown, GV-intact oocytes having larger UBE2I-containing bodies than transcriptionally active, meiotically incompetent growing oocytes. Inhibiting transcription in incompetent oocytes resulted in an increase in the size of the UBE2I-containing bodies. Overexpression of either wild-type UBE2I or catalytically inactive UBE2I resulted in an increase in the size of the UBE2I-containing bodies but also an increase in BrUTP incorporation, suggesting that transcriptional activation by UBE2I is independent of its catalytic activity. Although UBE2I-containing bodies did not completely colocalize with SUMO1 or SUMO2 and SUMO3, which were localized mainly on the nuclear membrane and in the nucleoplasm, UBE2I strikingly colocalized with SFRS2, which is a component of nuclear speckles and critical for mRNA processing. These results suggest a novel function for UBE2I and therefore sumoylation in gene expression..

Glial cell missing homolog 1 (GCM1) is an important transcription factor regulating placental cell fusion. Recently, we have demonstrated that GCM1 is a labile protein and that the F-box protein FBXW2 (F-box and WD repeat domain containing 2) mediates GCM1 ubiquitination for proteasomal degradation. Multiple factors are involved in the ubiquitin-proteasome degradation system. Therefore, in order to better understand the mechanism regulating GCM1 stability, we further isolated and characterized the E2 ubiquitin-conjugating enzyme responsible for FBXW2-mediated ubiquitination of GCM1 in this study. We prepared and screened a variety of E2 proteins in an in vitro ubiquitination assay system for GCM1 and found that UBE2D2 is required for the SCF^FBXW2 E3 ligase in regulation of GCM1 ubiquitination. We also demonstrated that the enzyme activity of UBE2D2 is required for GCMa ubiquitination and for association with the SCF^FBXW2 complex. Moreover, knocking down UBE2D2 expression by RNA interference not only suppressed FBXW2-mediated GCM1 ubiquitination, but also prolonged the half-life of GCM1 in vivo. Our results suggest that UBE2D2 is a functional E2 protein which, together with FBXW2, regulates GCM1 stability in the placenta..

During gametogenesis, germ cells must undergo meiosis in order to become viable haploid gametes. Successful completion of this process is dependent upon the expression of genes whose protein products function specifically in meiosis. Failure to express these genes in meiotic cells often results in infertility, whereas aberrant expression in somatic cells may lead to mitotic catastrophe. The mechanisms responsible for regulating the timely expression of meiosis-specific genes have not been fully elucidated. Here we demonstrate that E2F6, a member of the E2F family of transcription factors, is essential for the repression of the newly identified meiosis-specific gene, Slc25a31 (also known as Ant4, Aac4), in somatic cells. This discovery, along with previous studies, prompted us to investigate the role of E2F6 in the regulation of meiosis-specific genes in general. Interestingly, the core E2F6-binding element (TCCCGC) was highly conserved in the proximal promoter regions of 19 out of 24 (79.2%) meiosis-specific genes. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%). In the absence of E2F6, only a portion of these meiosis-specific genes was derepressed in somatic cells. However, endogenous E2F6 bound to the promoters of these meiosis-specific genes regardless of whether they required E2F6 for their repression in somatic cells. Further, E2F6 overexpression was capable of reducing their transcription. These findings indicate that E2F6 possesses a broad ability to bind to and regulate the meiosis-specific gene population..

The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5%. In conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species..

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

Pulsatile GnRH (GNRH) differentially regulates LH and FSH subunit genes, with faster frequencies favoring Lhb transcription and slower favoring Fshb. Various intracellular pathways mediate the effects of GNRH, including CaMK II (CAMK2), ERK, and JNK. We examined whether activation of these pathways is regulated by GNRH pulse frequency in vivo. GNRH-deficient rats received GNRH pulses (25 ng i.v. every 30 or 240 min for 8 h, vehicle to controls). Pituitaries were collected 5 min after the last pulse, bisected, and one half processed for RNA (to measure beta subunit primary transcripts [PTs]) and the other for protein. Phosphorylated CAMK2 (phospho-CAMK2), ERK (mitogen-activated protein kinase 1/3 [MAPK1/3], also known as p42 ERK2 and p44 ERK1, respectively), and JNK (MAPK8/9, also known as p46 JNK1 and p54 JNK2, respectively) were determined by Western blotting. The 30-min pulses maximally stimulated Lhb PT (8-fold), whereas 240 min was optimal for Fshb PT (3-fold increase). Both GNRH pulse frequencies increased phospho-CAMK2 4-fold. Activation of MAPK1/3 was stimulated by both 30- and 240-min pulses, but phosphorylation of MAPK3 was significantly greater following slower GNRH pulses (240 min: 4-fold, 30 min: 2-fold). MAPK8/9 activation was unchanged by pulsatile GNRH in this paradigm, but as previous results showed that GNRH-induced activation of MAPK8/9 is delayed, 5 min after GNRH may not be optimal to observe MAPK8/9 activation. These data show that CAMK2 is activated by GNRH, but not in a frequency-dependant manner, whereas MAPK3 is maximally stimulated by slow-frequency GNRH pulses. Thus, the ERK response to slow pulse frequency is part of the mechanisms mediating Fhb transcriptional responses to GNRH..

The amnion plays an important role during pregnancy and parturition. Though referred to as a single structure, this fetal tissue is regionally divided into placental amnion, reflected amnion, and umbilical amnion. Histological differences between placental amnion and reflected amnion led us to hypothesize that the amnion is biologically heterogeneous. The gene expression profiles of placental amnion and reflected amnion were compared in patients at term with no labor (TNL; n = 10) and in labor (TIL; n = 10). Real-time quantitative RT-PCR revealed a higher expression of IL1B mRNA in reflected amnion than in placental amnion in TNL cases but not in TIL cases. Extended screening using microarrays showed differential expression of 17 genes in labor, regardless of the region. Interestingly, 839 genes were differentially expressed between placental amnion and reflected amnion. Pathway analysis identified 19 signaling pathways, such as mitogen-activated protein kinase and transforming growth factor beta pathways, associated with region. Lipopolysaccharide (LPS) treatment of the amnion explants showed more robust activation of mitogen-activated protein kinase 3/1 (extracellular signal-regulated kinase 1/2) in placental amnion of TNL but not in TIL cases. Placental amnion from TNL and TIL cases showed a significant difference in the amplitude of IL1B mRNA induction by LPS. We report that the anatomical region has a substantial impact on the transcriptional program and the biological properties of the amnion. Labor-associated switching to a proinflammatory signature is a feature particular to placental amnion. The novel observations herein strongly suggest that the seemingly homogeneous amnion is biologically heterogeneous and compartmentalized, with implications for the physiology of pregnancy and parturition..

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH testosterone increased diploid germ cell survival during the first week, whereas only FSH testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

Ovine placentomes vary in shape, with type A placentomes being concave, type D convex, and types B and C intermediate in morphology. It has been speculated that as placentomes advance in type they differ in vascularity and nutrient transport capacity. Our objective was to determine cellularity and vascularity measurements, angiogenic factor expression, and arterial vasoactivity within different morphologic types of placentomes. On Day 130 of gestation, placentomes were collected from multiparous ewes (n = 38) and were evaluated for size, cellularity estimates, angiogenic factor mRNA expression, capillary vascularity (capillary size, capillary surface density [CSD], capillary number density [CND], and capillary area density [CAD]), and vasoreactivity to potassium chloride and angiotensin II. The average weight and size of type A and B placentomes were less (P < 0.01) than those of type C and D placentomes. Placentome morphology did not affect (P ≥ 0.24) cotyledonary or caruncular cellularity estimates or percentage of cellular proliferation. Placentome morphology affected (P ≥ 0.41) neither caruncular CAD, CND, CSD, or capillary size nor cotyledonary CND, CSD, or capillary size. Cotyledonary CAD was increased (P < 0.01) in type B and D placentomes compared with type A placentomes. Furthermore, placentome type did not affect (P ≥ 0.06) angiogenic factor gene expression in the cotyledon or the caruncle. Size, but not morphologic type of placentome, was associated with greater caruncular artery contractility to potassium chloride and angiotensin II (P < 0.01 for both). Placentome size, but not morphologic type, may be important for vascularity and nutrient transfer in the placenta of the pregnant ewe..

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P₄) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P₄ treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P₄ induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development..

Protein kinase A (PRKACA) has been documented as a pivotal regulator in meiosis and mitosis arrest. Although our previous work has established that PRKACA regulates cell cycle progression of mouse fertilized eggs by inhibiting M-phase promoting factor (MPF), little is known about the intermediate factor between PRKACA and MPF in the mitotic cell cycle. In this study, we investigated the role of the PRKACA/CDC25B pathway on the early development of mouse fertilized eggs. Overexpression of unphosphorylatable CDC25B mutant (Cdc25b-S321A or Cdc25b-S229A/S321A) rapidly caused G₂-phase eggs to enter mitosis. Microinjection of either Cdc25b-WT or Cdc25b-S229A mRNA also promoted G₂/M transition, but much less efficiently than Cdc25b-S321A and Cdc25b-S229A/S321A. Moreover, mouse fertilized eggs overrode the G₂ arrest by microinjection of either Cdc25b-S321A or Cdc25b-S229A/S321A mRNA, which efficiently resulted in MPF activation by directly dephosphorylating CDC2A-Tyr15, despite culture under conditions that maintained exogenous dibutyryl cAMP. Using a highly specific antibody against phospho-Ser321 of CDC25B in Western blotting, we showed that CDC25B-Ser321 was phosphorylated at the G₁ and S phases, whereas Ser321 was dephosphorylated at the G₂ and M phases in vivo. Our findings identify CDC25B as a potential target of PRKACA and show that PRKACA regulates G₂/M transition by phosphorylating CDC25B-Ser321 but not CDC25B-Ser229 on the first mitotic division of mouse fertilized eggs..

Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation..