Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology

The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.     

Lectin-binding sites in isolated equine cumulus-oocyte complexes: Differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA₁₂₀, and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B₄, and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.     

Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes

Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment 1, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum.     

Interaction of the EGFR signaling pathway with porcine cumulus oocyte complexes and oviduct cells in a coculture system

It has become increasingly recognized that coculture has a beneficial effect on the in vitro maturation (IVM) of oocytes and embryo development in many species. However, these effects of coculture on IVM have been documented only for their positive conditioning roles without any evidence on the precise mechanisms underlying the action of coculture systems on the development of cumulus oocyte complexes (COCs). It has been suggested that the epidermal growth factor receptor (EGFR) signaling pathway is important for development of COCs, mediated by several epidermal growth factor (EGF)-like proteins with downstream mitogen-activated protein kinase 1/3 signaling. Therefore, we hypothesized that canine oviduct cells (OCs) in a coculture system, which shows improvement of oocyte quality in several species, are associated with EGFR signaling by exposure to progesterone (P4; imitating its production before ovulation and its continuous increase while oocytes reside in the oviduct to complete maturation in dogs). We designed three experimental groups: control, OCs coculture exposed to P4, and OCs coculture without exposure to P4. The result showed that the OCs coculture exposed to P4 strongly expressed EGF-like proteins and significantly improved COCs and subsequent embryo development. Furthermore, the expression of EGFR-related genes in cumulus cells and GDF9 and BMP15 in oocytes was upregulated in the P4-treated group. This study provides the first evidence that OCs exposed to P4 can induce strong expression of EGF-like proteins, and OCs effectively mediate improved porcine COCs development and subsequent embryo development by altering EGFR signaling related mRNA expression.     

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.     

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short time priming of pig cumulus-oocyte complexes with FSH and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50-200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 microM) and FSH (100 U/L) priming CEO could time-dependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosis-activating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.     

Acoustic overexposure increases the expression of VGLUT-2 mediated projections from the lateral vestibular nucleus to the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) is a first relay of the central auditory system as well as a site for integration of multimodal information. Vesicular glutamate transporters VGLUT-1 and VGLUT-2 selectively package glutamate into synaptic vesicles and are found to have different patterns of organization in the DCN. Whereas auditory nerve fibers predominantly co-label with VGLUT-1, somatosensory inputs predominantly co-label with VGLUT-2. Here, we used retrograde and anterograde transport of fluorescent conjugated dextran amine (DA) to demonstrate that the lateral vestibular nucleus (LVN) exhibits ipsilateral projections to both fusiform and deep layers of the rat DCN. Stimulating the LVN induced glutamatergic synaptic currents in fusiform cells and granule cell interneurones. We combined the dextran amine neuronal tracing method with immunohistochemistry and showed that labeled projections from the LVN are co-labeled with VGLUT-2 by contrast to VGLUT-1. Wistar rats were exposed to a loud single tone (15 kHz, 110 dB SPL) for 6 hours. Five days after acoustic overexposure, the level of expression of VGLUT-1 in the DCN was decreased whereas the level of expression of VGLUT-2 in the DCN was increased including terminals originating from the LVN. VGLUT-2 mediated projections from the LVN to the DCN are likely to play a role in the head position in response to sound. Amplification of VGLUT-2 expression after acoustic overexposure could be a compensatory mechanism from vestibular inputs in response to hearing loss and to a decrease of VGLUT-1 expression from auditory nerve fibers.     

Increasing progesterone secretion and 3β-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P₄) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P₄ secretion in culture. In the G-L cells, P₄ secretion and percentage of cells manifesting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity increased concurrently with the period of culture. In the hCC, however, P₄ secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3β-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P₄ accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0–3 and 3–5 of culture, respectively, as compared to P₄ accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3β-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P₄ accumulation and percentage of 3β-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P₄ secretion are correlated and synchronous.     

Imp associates with squid and Hrp48 and contributes to localized expression of gurken in the oocyte

Localization and translational control of Drosophila melanogaster gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. Imp, the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation.     

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

The strength of negative plant–soil feedback increases from the intraspecific to the interspecific and the functional group level

One of the processes that may play a key role in plant species coexistence and ecosystem functioning is plant–soil feedback, the effect of plants on associated soil communities and the resulting feedback on plant performance. Plant–soil feedback at the interspecific level (comparing growth on own soil with growth on soil from different species) has been studied extensively, while plant–soil feedback at the intraspecific level (comparing growth on own soil with growth on soil from different accessions within a species) has only recently gained attention. Very few studies have investigated the direction and strength of feedback among different taxonomic levels, and initial results have been inconclusive, discussing phylogeny, and morphology as possible determinants. To test our hypotheses that the strength of negative feedback on plant performance increases with increasing taxonomic level and that this relationship is explained by morphological similarities, we conducted a greenhouse experiment using species assigned to three taxonomic levels (intraspecific, interspecific, and functional group level). We measured certain fitness‐related aboveground traits and used them along literature‐derived traits to determine the influence of morphological similarities on the strength and direction of the feedback. We found that the average strength of negative feedback increased from the intraspecific over the interspecific to the functional group level. However, individual accessions and species differed in the direction and strength of the feedback. None of our results could be explained by morphological dissimilarities or individual traits. Synthesis. Our results indicate that negative plant–soil feedback is stronger if the involved plants belong to more distantly related species. We conclude that the taxonomic level is an important factor in the maintenance of plant coexistence with plant–soil feedback as a potential stabilizing mechanism and should be addressed explicitly in coexistence research, while the traits considered here seem to play a minor role.     

Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology

The objective of the present study was to investigate the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus (SS) to regions of follicular retention in the cortex. The migration of horseradish peroxidase (HRP), used as a histochemically identifiable antigen, was followed by light and electron microscopy in C3H mouse popliteal lymph nodes obtained 1, 5, 15, and 30 min, and 5 and 24 hr after hindfoot pad injection of HRP. The observations showed that as early as 1 min after HRP injection, localization of antigen occurred at distinct sites in the SS and subjacent areas of the cortex on the afferent side. At these sites, between 1 min and 24 hr, the antigen formed light microscopically identifiable trails, which reached progressively deeper into the cortex with time toward individual follicular regions. By 24 hr this apparent migration of antigen was complete, and HRP was localized in follicles. This migration pattern did not occur on the efferent sides of lymph nodes, and it was dependent on the systemic presence of specific antibodies since it was observable only in passively immunized but not in nonimmune mice. Temporary retention of antigen by typical macrophages was also observed in the SS on the efferent side. This was minimal in nonimmune mice and was significantly enhanced in passively immunized mice. Electron microscopy indicated that the apparent migration of immune complexes was mediated by a group of cells observed in the migration path that had immune complexes sequestered on their surface or in plasma membrane infoldings. These antigen transporting cells (ATC) were relatively large nonphagocytic cells, with lobated or irregular euchromatic nuclei and cell processes of various complexity. ATC observed in or near the SS appeared to be less differentiated, were monocyte-like, and resembled non-Birbeck granule-containing Langerhans cell precursors or veiled cells. Others, located deeper in the cortex, appeared more differentiated, interdigitated with antigen-retaining dendritic cells, and shared morphologic characteristics with follicular dendritic cells (FDC). The results support the concepts that immune complexes are trapped in the SS and are transported by a group of non-phagocytic cells, other than lymphocytes, to follicular regions. The mechanism of transport may involve the migration of ATC with a concomitant maturation into FDC, or by a mechanism of ATC to FDC transport utilizing dendritic cell processes and membrane fluidity, or by a combination of the two mechanisms.     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Ovarian response and embryo production in llamas treated with equine chorionic gonadotropin alone or with a progestin-releasing vaginal sponge at the time of follicular wave emergence

The objective of the study was to compare the ovulatory response and embryo production in llamas (Lama glama) treated with a single dose of equine chorionic gonadotropin (eCG) alone or combined with intravaginal medroxyprogesterone acetate (MPA) at the time of follicular wave emergence. Llamas with a growing follicle ≥7 mm in diameter were assigned to one of the following groups: (1) Control (n = 28): Nonstimulated llamas were mated and embryos were collected 7 d after mating. (2) eCG (n = 32): Llamas were given 5 mg luteinizing hormone (LH) (Day 0) to induce ovulation, 1000 IU eCG on Day 2, a luteolytic dose of prostaglandin F_2α on Day 6, mating on Day 7, and embryo collection on Day 14. (3) eCG+MPA (n = 34): Llamas were treated as those in the eCG group, but a sponge containing 60 mg MPA was placed intravaginally from Days 2 to 6. Llamas that did not respond to synchronization or superstimulation were excluded, leaving data from n = 26, 26, and 27 in the control, eCG, and eCG+MPA groups, respectively, for statistical analysis. The mean (±SD) number of follicles > 7 mm at the time of mating was greatest in the eCG group, intermediate in the eCG+MPA group, and lowest in the control group (16.6 ± 5.3, 12.9 ± 3.7, and 1.0 ± 0.0, respectively, P < 0.001). The number of corpora lutea was similar between eCG and eCG+MPA groups (10.1 ± 2.9 and 8.6 ± 3.7, respectively); both were higher (P < 0.001) than in controls (0.9 ± 0.3). The number of embryos did not differ significantly between the eCG and eCG+MPA groups (4.8 ± 2.8 and 3.5 ± 3.0, respectively), but both were higher (P < 0.001) than in the controls (0.7 ± 0.4). In conclusion, eCG, with or without MPA effectively induced a superovulatory response and multiple embryo production in llamas.