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.

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 ∼10 min, remains above baseline for another ∼10 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.

Intraperitoneal injection of kisspeptin-54 induces a surge-like release of luteinizing hormone that stimulates ovulation in mice.

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, 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.

Luteinizing hormone (LH) induces ovulation by initiating signaling by the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how LH activation of its receptor (LHR) 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 LHR-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The proportion of LHR-expressing cell bodies in the inner half of the mural wall increases until the time of ovulation, with no change in the total number of cells expressing the receptor. Many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Following the ingression of the LHR-expressing cells, but still hours before ovulation, the follicular wall develops numerous invaginations and constrictions. LH stimulation of granulosa cell ingression may contribute to changes in the follicular structure that enable ovulation.

Jersey embryos have high lipid content and poor cryotolerance. High lipid and reactive oxygen species concentrations are associated with poor post-thaw survival and increased post-thaw apoptosis. It was hypothesized that culturing embryos in SOF-based medium (SCF1; SOF for conventional freezing will decrease lipid content, and adding l-ascorbic acid (l-AA) to freezing media will increase cryotolerance and decrease post-thaw apoptosis. A 2 × 2 factorial design was used to compare SOF v. SCF1 and additives in freezing media (control v. L-AA). In vitro-produced blastocysts were produced in 5 replicates by aspirating oocytes (n = 975) from 2 to 8 mm follicles of abattoir ovaries, maturing for 23 h, fertilizing with semen from 1 of 2 bulls, and culturing in SOF medium or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Randomly selected Day 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) for mitochondrial polarity. Remaining blastocysts were placed in 0.6 M sucrose in holding media for 2 min followed by equilibration in 1.5 M ethylene glycol and 0.5 M sucrose in holding media for 10 min with 0 or 0.1 mM l-AA. Blastocysts were thawed and assessed for re-expansion at 24 and 48 h, then stained with 4′,6-diamidino-2-phenylindole and a TUNEL assay to measure apoptosis. Ten images per stained blastocyst were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Image Pro software was used to measure fluorescence of Nile Red and Mitotracker, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by 1-way ANOVA and means were separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) by 2 (0 and 0.1 mM l-AA) and means were separated by Tukey’s HSD. Results (Table 1) indicate SCF1 increased blastocyst rate and post-thaw survival and decreased lipid content (P < 0.01) with no effect on mitochondrial polarity. Post-thaw, l-AA (Table 1) increased survival (P < 0.05) but had no effect on apoptosis. The SCF1 medium increases development and lowers lipid content, whereas l-AA may lower reactive oxygen species to increase cryotolerance. Table 1. Effect of media on development, lipid content, and mitochondrial polarity (top part), and of media and l-AA on post-thaw survival and apoptosis (lower part)

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

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