RESUMO
Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.
Assuntos
Hormônio Luteinizante , Receptores do LH , Feminino , Camundongos , Animais , Hormônio Luteinizante/metabolismo , Receptores do LH/metabolismo , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Ovulação/fisiologia , Mamíferos/metabolismoRESUMO
Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 minutes of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 hours after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 hours after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.
RESUMO
In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, and then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.
In vitro produced (IVP) bovine embryos suffer from several physiological abnormalities that interfere with their ability to withstand the freezing process, a vital step in shipping and distribution of IVP embryos. To overcome these challenges, we performed a series of experiments to determine the optimal culture medium to best support the developing embryo. This new in vitro embryo culture medium is referred to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1). The medium is supplemented with various factors to more closely mimic the uterine environment, improve mitochondrial function, and decrease lipid accumulation. The results show that IVP embryos cultured in SCF1, slow frozen using an optimized technique, and transferred into recipients have a pregnancy rate that is similar to non-frozen IVP embryos. These findings suggest that SCF1 improves developmental competence of bovine IVP embryos and their ability to withstand cryopreservation, which can improve pregnancy rates and efficiency of assisted fertility operations within the dairy cattle industry.
Assuntos
Criopreservação , Transferência Embrionária , Animais , Blastocisto , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , Congelamento , Gravidez , Taxa de GravidezRESUMO
Meiotic arrest and resumption in mammalian oocytes are regulated by 2 opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic guanosine 5'-monophosphate (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains incompletely understood, due in part to imprecise information about the cellular localization of these 2 proteins. To investigate their localization, we generated mouse lines in which hemagglutinin epitope tags were added to the endogenous LHR and NPR2 proteins, and used immunofluorescence and immunogold microscopy to localize these proteins with high resolution. The results showed that the LHR protein is absent from the cumulus cells and inner mural granulosa cells, and is present in only 13% to 48% of the outer mural granulosa cells. In contrast, NPR2 is present throughout the follicle, and is more concentrated in the cumulus cells. Less than 20% of the NPR2 is in the same cells that express the LHR. These results suggest that to account for the LH-induced inactivation of NPR2, LHR-expressing cells send a signal that inactivates NPR2 in neighboring cells that do not express the LHR. An inhibitor of gap junction permeability attenuates the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with this mechanism contributing to how NPR2 is inactivated in cells that do not express the LHR.
Assuntos
GMP Cíclico/metabolismo , Folículo Ovariano/enzimologia , Receptores do Fator Natriurético Atrial/metabolismo , Receptores do LH/metabolismo , Animais , Feminino , Camundongos , Microscopia Eletrônica de Varredura , Folículo Ovariano/ultraestruturaRESUMO
California condors released in costal sites are exposed to high levels of xenoestrogens, particularly p,p'-DDE, through scavenging of marine mammal carcasses. As a result, coastal condors carry a higher contaminant loads and experience eggshell thinning when compared to their inland counterparts. Given that condor estrogen receptors (Esrs) are activated by physiologically relevant levels of xenoestrogens, differences in vulnerability to endocrine disruption may exist depending on which Esr variant(s) an individual condor possesses. This work aims to characterize genetic polymorphisms in estrogen receptor genes (ESRs) in California condors; one identified for condor estrogen receptor 1 (ESR1) (N161S, E162D) and one in the ESR2 (T114M) gene. Each variant was confirmed in individual founder birds by direct PCR sequencing as well as in first generation offspring to understand the introduction of the alleles into the pedigree (6 birds for ESR1 and 5 birds for ESR2). Site-directed mutagenesis was performed on wild type receptors to produce each of the full-length ESR variants and activation of Esr1 and Esr2 variant and wild type receptors by xenoestrogens was compared. Maximal activation of the variant form of Esr1 was significantly higher (p < 0.05) in response to ethinyl estradiol (EE2), o,p'-DDE, p,p'-DDE, p,p'-DDT and p,p'-DDD compared to wild type Esr1. For Esr2 the wild type maximal activation was higher in response to o,p'-DDE, p,p'-DDE, o,p'-DDT, and p,p'-DDT. Although significant differences in activation of condor Esr variants by xenoestrogens occurred at high (micromolar) concentrations, they correspond to circulating concentrations previously reported in coastal birds. Release and relocation of California condors to the coast is a promising avenue for recovery, however, reproductive problems associated with xenoestrogen exposure pose a sub-lethal threat to long-term success. Based on above findings, future release decisions could be informed by ESR form(s) individual birds possess to reduce deleterious effects of xenoestrogen exposure and ultimately improve reproductive success in wild populations.
Assuntos
Fitoestrógenos/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Aves , Feminino , MasculinoRESUMO
In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at â¼10 min, remains above baseline for another â¼10 min, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 h and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does delay the time to 50% ovulation by about 3 h. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.