Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA).

The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.

Effect of semen collection in extender solution on the characteristics of goat spermatozoa.

The objective of this experiment was to investigate whether the motility parameters and acrosome integrity of goat ejaculated spermatozoa are affected by collecting semen into tubes containing an extender, and thereby determine the significance of reducing contact between seminal plasma and the sperm membrane at ejaculation. Semen were collected from three goats into tubes containing 0, 1 or 10 ml extender, or collected into tubes containing 10 ml extender supplemented with 0.1, 1 or 5% BSA. Sperm motion parameters were evaluated immediately after collection, after washing, and during a 3-h thermal resistance test. Acrosome integrity was assessed using FITC-PNA staining. Semen collection into tubes containing 10 ml extender produced higher sperm motility, progressive motility, and acrosome integrity than that using a smaller volume of extender. Furthermore, collection into 5% BSA-containing extender exhibited higher sperm characteristics and maintained high sperm motility and progressive motility throughout incubation. In conclusion, semen collection into tubes with a large volume of extender, especially extender containing higher concentrations of BSA, improved the quality of ejaculated spermatozoa, strongly suggesting that the in vitro functional characteristics of the spermatozoa were abruptly modified by flash sperm contact with accessory sex gland fluid at ejaculation.

Studies of the role of steroid hormone in the regulation of oocyte maturation in cattle.

BACKGROUND: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor. METHODS: In experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion. RESULTS: Experiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion. CONCLUSION: Our results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion.

Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation.

BACKGROUND: The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. METHODS: First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 degrees C to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4 h during maturation was determined using the developed method. RESULTS: The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/microL DNase I with 10 U/microL for 30 min, an Mg concentration of 1.5 mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8 h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. CONCLUSION: It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction.

Studies on substantially increased proteins in follicular fluid of bovine ovarian follicular cysts using 2-D PAGE and MALDI-TOF MS.

BACKGROUND: The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC). METHODS: Proteins in normal and cystic FF samples were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were compared using silver stained gel images with PDQuest image analysis software. Peptides from these increased spots were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and were identified based on the NCBI database by a peptide mass fingerprinting method. RESULTS: Comparative proteomic analysis showed 8 increased protein spots present in cystic FF. MS analysis and database searching revealed that the increased proteins in cystic FF were bovine mitochondrial f1-atpase (BMFA), erythroid associated factor (EAF), methionine synthase (MeS), VEGF-receptor, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (HSP70), beta-lactoglobulin (BLG) and succinate dehydrogenase Ip subunit (SD). CONCLUSION: Our results suggest that these proteins are overexpressed in BOFC, and that they may play important roles in the pathogenesis of BOFC. Furthermore, these proteins in the FF could be useful biomarkers for BOFC.

Gonadotropin-induced delta14-reductase and delta7-reductase gene expression in cumulus cells during meiotic resumption of porcine oocytes.

Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of Delta14-reductase and Delta7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both Delta14-reductase mRNA and Delta7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either Delta14-reductase inhibitor, AY9944-A-7, or Delta7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 microM AY9944-A-7 or 20 microM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of Delta14-reductase and Delta7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa.

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality = 370, pH = 7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035-0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved (P < 0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0 +/- 1.5% and 65.0 +/- 1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest (P < 0.05) when 0.05% (74.0 +/- 1.1) and 0.075% (70.0 +/- 1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 degrees C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.

Mitogen-activated protein kinase kinase inhibitor suppresses cyclin B1 synthesis and reactivation of p34cdc2 kinase, which improves pronuclear formation rate in matured porcine oocytes activated by Ca2+ ionophore.

To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 microM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-microM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca(2+)-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.

Effect of protein kinase C activator on mitogen-activated protein kinase and p34(cdc2) kinase activity during parthenogenetic activation of porcine oocytes by calcium ionophore.

The objective of this study was to elucidate the role of a [Ca2+]i rise and protein kinase C (PKC) activation on decreases of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase activity during parthenogenetic activation of porcine oocytes. In oocytes treated with 50 microM Ca2+ ionophore, degradations of both p34(cdc2) kinase and MAP kinase activity were observed and half of these oocytes formed pronuclei. However, a supplement of PKC inhibitor, calphostin C, after 50 microM Ca2+ ionophore treatment, was sufficient to inhibit the inactivation of MAP kinase and pronuclear formation in the oocytes. These results showed that PKC played an important role in Ca2+-induced oocyte activation. On the other hand, 10 microM Ca2+ ionophore treatment could not affect the MAP kinase activity but induced a transient decrease of p34(cdc2) kinase activity, which resulted in recovery of p34(cdc2) kinase activity and progression to meiotic metaphase III stage. To investigate the effects of PKC activator on oocytes treated with 10 microM Ca2+ ionophore, matured oocytes were cultured with phorbol 12-myriatate 13-acetate (PMA), after 10 microM Ca2+ ionophore treatment. The additional treatment suppressed the recovery of p34(cdc2) kinase activity and rapidly induced a decrease of MAP kinase activity, and these low activities were maintained until 12-h cultivation. As a result, a significantly higher percentage of these oocytes (67%) had pronuclei at 12-h cultivation. Moreover, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not p34(cdc2) kinase activity in matured porcine oocytes. In conclusion, the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34(cdc2) kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes.

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes.

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.

FSH and LH induce progesterone production and progesterone receptor synthesis in cumulus cells: a requirement for meiotic resumption in porcine oocytes.

The aim of this study was to investigate the role of progesterone in the meiotic resumption of porcine oocytes. Progesterone production and progesterone receptor (PR) immunoreactivity in cumulus cells were not detected in porcine cumulus-oocyte complexes (COC) when observations were made either just after collection from the follicles or after 28 h cultivation without LH and FSH. However, the addition of LH and FSH induced PR expression in cumulus cells, concomitant with increased progesterone production. To assess the role of progesterone in the COC, an inhibitor of progesterone production, aminoglutethimide (AGT), was administered. The addition of AGT to the medium with LH and FSH significantly suppressed progesterone production in a dose-dependent fashion. When COC were cultured with LH, FSH and 0.5 x 10(-3) mol/l AGT, almost complete inhibition of progesterone production and of germinal vesicle breakdown (GVBD) was seen. However, this inhibitory effect on GVBD was overcome by additional progesterone. Moreover, 0.5 x 10(-3) mol/l AGT also suppressed the reduction in connexin43, a gap junctional protein, in cumulus cells after 28 h cultivation, and increased the level of cyclic AMP in oocytes. These results support the hypothesis that the binding of progesterone, which was secreted by LH- and FSH-stimulated cumulus cells, to its newly synthesized receptor induces GVBD in porcine oocytes, possibly through a reduction of connexin43 in cumulus cells.

Roles of cAMP in regulation of both MAP kinase and p34(cdc2) kinase activity during meiotic progression, especially beyond the MI stage.

Time-dependent changes in the level of adenosine cyclic AMP (cAMP) in porcine oocytes during meiotic progression from the germinal vesicle stage (GV stage) to the metaphase II stage (MII stage) were examined using reversed-phase HPLC with UV detection. The concentration of cAMP in oocytes reached a peak at 8 hr of cultivation of cumulus-oocyte complexes (COCs), but it was dramatically decreased after 12-hr cultivation. After a 28-hr cultivation period, the level of cAMP in the oocytes had significantly reduced further, and the basal level of cAMP was observed in oocytes cultured at 32 hr and for up to 48 hr. When phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase C (PKC) in cumulus cells [which were required for meiotic progression to the MII stage in porcine oocytes (Shimada and Terada, 2001: Biol Reprod 64:1106-1114)] was suppressed by each specific inhibitor following initial 24-hr cultivation of COCs, cAMP level in the oocytes was significantly increased. After 24-hr cultivation in the maturation medium, COCs, which were cultured for an additional 24 hr in the presence of either forskolin or 3-isobutyl-1-methylxanthine (IBMX), exhibited a significant increase in the oocyte cAMP level to the similar level of that in oocytes cultured with PI 3-kinase inhibitor or PKC inhibitor, and the addition of each agent significantly suppressed meiotic progression from the MI to the MII stage and the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) kinase. These results demonstrated that when transported into oocytes from the cumulus cells via gap junctions, cAMP plays an important role not only in meiotic resumption, but also in the regulation of meiotic progression beyond the MI stage in porcine oocytes.