Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro.

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Responses of the estrous cycle in dairy cows exposed to electric and magnetic fields (60 Hz) during 8-h photoperiods.

To study the effects of exposure to extremely low frequency (ELF) electric and magnetic fields (EMF) on the estrous cycle of dairy cows under short-day photoperiod, 16 non-lactating, non-pregnant Holstein cows were exposed to a vertical electric field of 10 kV/m and a horizontal magnetic field of 30 microT for 16 h per day in a cross-over design consisting of two sequences. Each sequence included three periods, and each period corresponded to the duration of one estrous cycle. All animals were maintained under short photoperiod (8 h light/16 h dark) during the trial. Exposure to EMF had an impact on the duration of a complete estrous cycle (P<0.01) and on the duration of the luteal phase (P<0.01). The mean duration of one cycle was 19.5+/-0.4 for the control and 21.3+/-0.4 days for the exposed animals, respectively. The mean duration of the luteal phase was 15.4+/-0.4 days for the control and 17.2+/-0.4 days for the exposed group. The total area under the progesterone (P(4)) curve, the amplitude of the curve or the slope of the P(4) rise at the onset of the luteal phase were not affected by EMF exposure. Results indicate that exposure to EMF may increase the duration of the estrous cycle.

Seasonal variation in preovulatory events associated with synchronization of estrus in dwarf goats.

This experiment was conducted to define the temporal relationships among estrus, the LH surge and ovulation after estrus synchronization in dwarf goats and to assess the effect of season on these parameters. In November (breeding season), March (transition period) and July (non-breeding season), estrus was synchronized in 12 dwarf goats by means of intravaginal sponges containing 60 mg medroxyprogesterone acetate (MAP) for 10 d, coupled with 125 microg cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A different group of animals was used during each time period. Onset of estrus was monitored using two males, and blood samples for the measurement of plasma LH were collected at 2-h intervals from 24 to 60 h after sponge removal. Ovulation was confirmed by laparoscopy at 54 and 72 h after sponge removal. A seasonal shift was detected in the intervals to onset of estrus, LH surge, and ovulation after sponge removal (P<0.05), with sponge removal to onset of estrus being shorter (P<0.05) in November (25.0 +/- 1.56 h) and July (28.9 +/- 2.43 h) than in March (40.9 +/- 3.27 h). The intervals between onset of estrus and the LH surge and between the LH surge and ovulation were found to be constant throughout the different seasons. An optimal time for breeding, artificial insemination, oocyte and embryo recovery, and embryo transfer may be predicted using information gained from these studies.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Formation and early development of the corpus luteum in pigs.

Numerous corpora lutea form from the multiple follicles that ovulate during the oestrous cycle of pigs. Vascular elements invade the follicle from the theca compartment, first centripetally, and subsequently by lateral branching of centripetal veins and arteries. The vessels are the vehicle for dispersion of steroidogenic theca cells throughout the corpus luteum. Mitosis occurs in both the theca and granulosa layers before ovulation, and in luteal cells well into the luteal phase. Luteal cell proliferation undergoes gradual restriction as the corpus luteum matures, but the mechanisms of exit from the cell cycle are unknown. The extracellular ligands that direct luteinization and maintain the corpus luteum include LH, prolactin, insulin and insulin-like growth factors (IGFs). These ligands induce qualitative and quantitative changes in steroid output, with progesterone as the principal product. These changes upregulate the cholesterol synthetic pathways to increase substrate availability. The intracellular regulation of luteinization is complex. A model is presented in which LH stimulates arachidonic and lineoleic acid metabolism to produce ligands for the nuclear proteins of the peripheral peroxisome activator receptor family. These ligands have downstream effects on cell differentiation and exit from the cell cycle. Luteal function is maintained by interactions among ligands, cholesterol regulatory proteins and constitutively expressed and regulated transcription factors.

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro.

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL(-1) FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen-thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 microM calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (10.8% v 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca(2+)-ionophore. However, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10000g and stimulation with calcium ionophore A23187 than in the control (18.4% v 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be < or = 7000g to enhance the visibility of nuclear elements for further micromanipulation.

Porcine leptin receptor: molecular structure and expression in the ovary.

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT-PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation.

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI).

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation.

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F2 alpha synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F2 alpha were lower (P < 0.01) than controls at 40 h and ovulation rate was suppressed (P < 0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P < 0.01), but the levels of 15-HETE and prostaglandin F2 alpha were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.

Connexin 43 gap junction protein expression during follicular development in the porcine ovary.

Connexin 43, a member of the highly conserved connexin family of gap junction proteins, is expressed in the pig ovary. In other species, ovarian connexin 43 expression and phosphorylation are hormonally regulated. We characterized connexin 43 expression and phosphorylation in the ovaries of mature pigs during the estrous cycle and in prepubertal gilts during follicular development induced by eCG (750 IU)/hCG (500 IU; 72 h later). Ovarian connexin 43 protein expression and phosphorylation were examined by immunoblot analysis. Connexin 43 was localized to specific follicular cell types during development by immunofluorescence. While no change in total connexin 43 protein expression was seen during the cycle, connexin 43 phosphorylation was significantly higher (p < 0.05) during the late follicular stage of the cycle than during the early luteal and early to mid-follicular stages. In ovaries of eCG/hCG-primed prepubertal pigs, connexin 43 protein levels remained steady, while phosphorylation of the protein increased significantly at 72 h and 84 h after eCG treatment (p < 0.05), then declined to pretreatment levels by 96 h (24 h post-hCG administration). Immunoreactive connexin 43 was localized predominantly to granulosa cells of cyclic pigs and eCG/hCG-primed prepubertal gilts. Follicular connexin 43 was highest between 60 h and 84 h after eCG and declined after hCG administration. Connexin 43 was not detected in morphologically atretic follicles, stroma, or vascular tissue of the ovary. This is the first evidence that porcine ovarian connexin 43 phosphorylation is differentially regulated during follicular development. The results suggest that hormonally induced changes in connexin 43 phosphorylation may play a coordinating role in porcine follicular development.

Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2).

A spontaneously established porcine granulosa cell line (PGC-2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)-treated prepubertal gilts. This established cell line has undergone approximately 100 passages and shows contact-inhibition of growth. PGC-2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast-derived. Immunoblotting revealed that PGC-2 expresses cadherin, an epithelial Ca+2-dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of approximately 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled-ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin (10 microM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified of SH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC-2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

Ultrastructural changes in bovine oocytes cryopreserved by vitrification.

Oocytes recovered from abattoir-derived ovaries were exposed to a cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% fetal calf serum in tissue culture medium 199) for less than 20 s and vitrified either at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed by fertilization and culture in vitro to the blastocyst stage. To identify ultrastructural changes, some of the vitrified oocytes that were morphologically normal after thawing were immediately processed for transmission electron microscopy after DAP213 removal. Cleavage rates for vitrified IVM oocytes were 4.5 and 6.7% using one-step and three-step cryoprotectant dilution procedures, respectively. Four (3%) oocytes developed to the eight-cell stage with the three-step procedure, but none formed blastocysts. None of the GV oocytes cleaved, while 66.7% (78/117) of controls developed to the two-cell stage and 19.2% (15/78) of those became blastocysts. Vitrification induced profound ultrastructural modifications in microvilli, mitochondria, vesicle formation, and the ooplasm of GV oocytes, whereas these structures were generally better preserved in IVM oocytes. The integrity of cell organelles was relatively better maintained following the three-step than after the one-step procedure in both GV and IVM oocytes. Changes in the zona pellucida (ZP) of IVM oocytes due to vitrification were associated with fewer cortical granules in the ooplasm. Since previous work showed that short-term exposure to DAP213 did not cause ZP alterations in IVM oocytes, these findings suggest that ZP damage due to low temperatures may result from the premature release of cortical granules.

In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution.

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoprotectant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P < .05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P < .05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that 1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and 2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.

Partial purification and characterization of a protein in porcine follicular fluid which restricts sperm-egg interaction in vitro.

An attempt was made to isolate and characterize a component in preovulatory porcine follicular fluid (pFF) which has a restricting effect on sperm-egg interaction in vitro. Using the zona-free hamster ova (eggs) penetration assay as an in vitro test system, it was shown previously that the numbers of porcine spermatozoa attached to or penetrated into each egg and the number of eggs with sperm attached or penetrated decreased significantly as the concentration of pFF was increased in the culture medium. In the present study, the component in pFF having these effects was shown to be a heat stable, nonsteroidal substance which retained its activity after dialysis, lyophilization and gel filtration chromatography. The activity was also found to be present in preovulatory homologous serum. Separation of the material on protein type gel filtration columns with detection at 280 nm, together with the banding seen with Coomassie staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggests that it is a protein. Based on high pressure liquid chromatographic separation (HPLC) and SDS-PAGE analyses, the bioactivity could be due to a single protein of 87 kD or to one or more of three smaller proteins, possibly disaggregated products of the 87 kD protein, in the range of 26-28 kD.

Morphological and functional characteristics of preovulatory follicles in large white and Meishan gilts.

Research was conducted to determine whether there are preovulatory follicular characteristics unique to Chinese Meishan (MS) sows that may contribute to their high prolificacy compared to that of European breeds. Follicles were recovered during the late follicular phase, following altrenogest withdrawal, from the ovaries of MS and Large White (LW) gilts before and after the administration of hCG given to mimic the LH surge. Following incubation of whole follicles for 1 h in vitro, media were collected for measurement of estradiol-17 beta, testosterone, and progesterone concentrations, and follicles were either fixed to assess number of granulosa and theca interna cells or cut into explants to test for aromatase activity over an additional 24-h incubation period. In MS gilts, follicles were smaller before and after hCG, although their growth was greater after hCG than was the growth of LW follicles. The LW and MS follicles contained relatively similar numbers of theca interna cells, whereas the numbers of granulosa cells in MS follicles were marginally (before hCG) or significantly (after hCG) less than those found in LW follicles. Before hCG, follicles of comparable size from both breeds produced similar amounts of estradiol and progesterone, whereas MS follicles produced less (P < .05) testosterone. Aromatase activity was not stimulated by FSH in either case, although a breed x follicle size interaction (P < .05) indicated a different pattern of aromatase activity between the breeds. After hCG, testosterone production was similar in MS and LW follicles, but estradiol (P < .05) and progesterone (P < .01) production were greater in MS follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of whole and demi-embryos of mice in culture and in vivo after supercooled storage.

Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at -5 degrees C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant (P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.

Developmental expression of the relaxin gene in the porcine corpus luteum.

Northern analysis and in-situ hybridization were used to follow the development of relaxin gene expression in the newly forming corpus luteum (CL) after ovulation and throughout luteal development. Alkaline phosphatase (AP) was used as a marker of theca-derived lutein cells and the relationship between AP-positive and relaxin mRNA-containing cells was assessed. Ovaries from prepubertal pigs treated with pregnant mares serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG) were collected during the periovulatory period and at various times during 19 days after ovulation. In addition, CL from cyclic pigs on days 10 and 16 were used to monitor relaxin gene expression in small and large luteal cells. Northern analysis revealed that relaxin gene expression increased with CL development in the PMSG/hCG-treated pig, reaching maximal levels at around day 14 post-ovulation. Thereafter, as the CL regressed, the level of relaxin mRNA declined. In CL from cyclic pigs at day 10 of the cycle, only small luteal cells expressed relaxin mRNA. However, by day 16 of the cycle, large luteal cells were the source of relaxin gene expression. In-situ hybridization studies revealed that in the early CL (up to 30 h post-ovulation), the relaxin gene transcript was observed in cells along the margins of the CL and in the core of the infolding follicle wall corresponding to the AP-positive, luteinized theca cell layer. As luteinization progressed, the theca and granulosa cell layers could no longer be distinguished morphologically (from 54 h after ovulation until day 9). However, the pattern of relaxin hybridization persisted along the periphery in bands of cells penetrating the CL, and coincided with areas of AP staining, indicating that the theca lutein cells were the site of relaxin gene expression. At day 14, relaxin hybridization and AP staining were distributed throughout the luteal tissue. With CL regression both AP staining and relaxin hybridization declined. This pattern of relaxin hybridization in the CL of the gonadotrophin-primed pig was identical to that observed in cyclic pigs on days 10 and 16 of the cycle. These findings indicate that theca interna cells retain their ability to express the relaxin gene following ovulation and luteinization. In the early CL, the small theca-derived lutein cells are the source of relaxin transcript. However, as the CL becomes fully differentiated, the large granulosa-derived lutein cells acquire the capacity to express the relaxin message.

Biological mimicry of gonadotropin action by antiidiotypic antibodies to luteinizing hormone: characterization and biological properties.

High affinity polyclonal rabbit antibodies to ovine (o) pituitary LH [anti-oLH-immunoglobulin G (IgG) Ab1] were used to immunize young Southdown lambs. Their serum samples as well as those from controls receiving normal rabbit IgG were studied for the presence of anti-Ab1 antibodies. In RIAs using [125I]oLH and affinity-purified Ab1, sera from experimental sheep showed high activity, as expressed in oLH equivalents. These sera also showed ability to compete with [125I]oLH for binding to receptor on pig ovarian and testicular membranes. The antiidiotypic antibodies (Ab2) in experimental sheep sera were purified by successive affinity chromatography on immobilized rabbit normal IgG and immobilized oLH-IgG columns. Ab2-IgG eluted from the latter mimicked oLH in RIAs and RRAs. These purified Ab2 antibodies were also of a stimulatory type, because they elicited progesterone production in rat granulosa cells and collagenase-dispersed rat Leydig cells. This stimulatory action was counteracted by coincubation with anti-oLH-IgG, which would also terminate (oLH) hormone action in a similar manner. The Ab2 antibodies had no effect on oFSH RIA or on the binding of [125I]oFSH to pig ovarian receptors, indicating specificity with respect to LH antigenic structure and function. As can be expected from the choice of the immunogen (polyclonal anti-oLH-IgG), only a small percentage of the true Ab2 population could display biological mimicry of the original antigen (oLH). Their presence in circulation during 6-8 months had no effect on testicular size or body growth. The formation of Ab2 antibodies to rabbit anti-oLH-IgG was also demonstrated in male rats, but these were not purified. In this instance also there was no effect on testicular weight after 6 months of immunization. These results show the feasibility of producing antiidiotypic antibodies that stimulate gonadal function in a manner much like the pituitary gonadotropin (oLH).

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.

The effect of supercooling on the developmental capacity of mouse embryos.

The effect of supercooled storage (at subzero temperatures without ice formation) on compacted mouse morulae and early blastocysts was studied. The embryos were equilibrated with one of three storage solutions containing 1, 3, or 6% each of methanol and glycerol and cooled to -2, -5, -10, or -15 degrees C and stored for up to 24 h to assess the effect of subzero storage at different temperatures and concentrations of the permeating cryoprotectants on embryo survival. Early blastocysts showed substantially greater survival than morulae and, in general, survival of embryos of either stage increased with the concentration of cryoprotectant, while the proportion of embryos surviving decreased with decreasing storage temperature and with increased duration of storage.