Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Feeding an enhanced diet to Holstein heifers during the preweaning period alters steroid receptor expression and increases cellular proliferation

Preweaning diet and estradiol treatment alters mammary development. Our objectives were to study the effects of diet and estradiol on proliferation of mammary epithelial cells and expression of estrogen receptor α (ESR1) and progesterone receptors (PGR) in these cells. Thirty-six Holstein heifer calves were raised on (1) a control milk replacer fed at 0.44 kg of powder/head per day, dry matter (DM) basis (restricted, R; 20.9% crude protein, 19.8% fat, DM basis), or (2) an enhanced milk replacer fed at 1.08 kg of powder/head per day, DM basis (Enhanced, EH; 28.9% crude protein, 26.2% fat, DM basis). Milk replacer was fed for 8 wk. At weaning, a subset (n = 6/diet) of calves were euthanized and had tissue harvested. Remaining calves received estradiol implants (E₂) or placebo and were euthanized at wk 10 to harvest tissue. Treatments were (1) R, (2) R + E₂ (R-E2), (3) EH, and (4) EH + E₂ (EH-E2). One day before euthanasia calves were given bromo-2′-deoxyuridine (BrdU; 5 mg/kg of body weight). At euthanization, mammary parenchyma was removed and fixed. Tissue sections from zone 1 (cisternal), 2 (medial), and 3 (distal) within the mammary gland were stained with hematoxylin and eosin and antibodies to measure expression of ESR1, PGR, and incorporation of BrdU. At wk 8, R-fed calves had more PGR-expressing cells in distal parenchyma; however, PGR expression intensity was greater in EH-fed calves. The proportion of cells expressing ESR1 was not affected by diet, but expression intensity (receptors per positive cell) was greater in EH-fed calves across all zones (62–81%). Overall, the percent BrdU-positive epithelial cells was 2 and 0.5 fold greater for EH-fed calves in zone 2 and 3. The proportion of labeled cells was greater in terminal ductal units than in subtending ducts, and treatment effects were more evident in terminal ductal units. At wk 10, calves treated with estradiol had 3.9-fold greater PGR expression intensity. The intensity and percent of cells expressing ESR1 was lowest in estradiol-treated calves. Overall, estradiol-treated calves had the greatest number of proliferating epithelial cells. Moreover, in zone 3, EH-E2 calves had a higher percentage of proliferating cells than in all other treatments. Results indicate both diet and estradiol administration alter proliferation rates of the mammary epithelium and that changes in expression of ESR1 and PGR are involved in enhanced mammary development. The data support our hypothesis that enhanced preweaning feeding increases the mammary tissue responsiveness to mammogenic stimulation.     

Effects of Staphylococcus aureus intramammary infection on the expression of estrogen receptor α and progesterone receptor in mammary glands of nonlactating cows administered estradiol and progesterone to stimulate mammary growth

Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to alter mammary structure and negatively affect the amount of mammary epithelium in the gland. Mechanisms responsible for the observed changes in mammary growth during an IMI are poorly understood, yet the importance of the key mammogenic hormones driving mammary growth is well recognized. This study's objective was to characterize the expression of estrogen receptor α (ESR1) and progesterone receptor (PGR) in mammary glands stimulated to grow and develop in the presence or absence of an IMI as well as preliminarily characterize myoepithelial cell response to IMI. Mammary growth was stimulated in 18 nonpregnant, nonlactating dairy cows using subcutaneous estradiol and progesterone injections, and 2 culture-negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) postchallenge and examined using immunofluorescence microscopy to quantify positive nuclei and characterize staining features. There tended to be a greater number of ESR1-positive nuclei observed across 8 random mammary parenchyma fields of view in saline quarters than in Staph. aureus quarters (201 vs. 163 ± 44 nuclei). Saline quarters also contained a greater number of PGR-positive nuclei (520 vs. 440 ± 45 nuclei) and myoepithelial cells (971 vs. 863 ± 48 nuclei) than Staph. aureus-challenged quarters. However, when ESR1, PGR, and myoepithelial nuclei counts were adjusted for Staph. aureus quarters containing less epithelium, differences between quarter treatments abated. The examined ESR1 and PGR staining characteristics were similar between saline and Staph. aureus quarters but were differentially affected by day of tissue collection. Additionally, nuclear staining area of myoepithelial cells was greater in Staph. aureus quarters than in saline quarters. These results indicate that IMI had little effect on the number or staining characteristics of ESR1- or PGR-positive nuclei relative to epithelial area, but myoepithelial cells appear to be affected by IMI and the associated inflammation in nonlactating mammary glands that were stimulated to grow rapidly using mammogenic hormones. Accordingly, reductions in mammary epithelium in affected glands are not suspected to be resultant of alterations in the number or staining characteristics of ESR1- or PGR-positive mammary epithelial cells.     

Immunohistochemical localization and expression of the hyaluronan receptor CD44 in the epithelium of the pig oviduct during oestrus

Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in relation to spontaneous ovulation and the presence of spermatozoa indicate that the hyaluronan CD44-signalling pathway may play a role in oviduct function during sperm storage and fertilization in pigs.     

Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares

Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation. Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer. Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing. The percentage of oocytes resulting in embryonic vesicles was nearly identical (P >0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%). More (P <0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer. Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P <0.05), although addition of hormones and growth factors during culture did not improve (P >0.05) development of embryos.     

Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce

Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.     

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.     

Expression of selenium-binding protein 1 characterizes intestinal cell maturation and predicts survival for patients with colorectal cancer

To identify candidate genes involved in the development of colorectal cancer, we used cDNA microarrays to analyze gene expression differences between human colorectal tumors and paired adjacent normal mucosa. We identified approximately 3.5-fold significant downregulation of selenium-binding protein 1 (SBP1) in colorectal tumors compared to normal mucosa (p = 0.003). Importantly, stage III colorectal cancer patients with low tumor-SBP1 expression had significantly shorter disease-free and overall survival as compared with those patients with high tumor-SBP1 expression (p = 0.04 and 0.03, respectively). We further characterized the role of SBP1 in colorectal cancer in vivo and in vitro. In normal tissue, SBP1 was maximally expressed in terminally differentiated epithelial cells on the luminal surface of crypts in the large intestine. Consistent with this in vivo localization, SBP1 was upregulated during in vitro colonic cell differentiation along the absorptive (Caco-2) and secretory (HT29 Clones 16E and 19A) cell lineages. Downregulation (approximately 50%) of SBP1 expression by small interfering RNA in colonic cancer cells was associated with reduced expression of another epithelial differentiation marker, carcinoembryonic antigen (CEA), although PCNA and p21(WAF1/cip1 )expression were not altered. These data demonstrate that higher expression of SBP1 is associated with differentiation of the normal colonic epithelia and may be a positive prognostic factor for survival in stage III colorectal carcinoma.     

Effect of ectopic expression of homeoprotein EGAM1C on the cell morphology, growth, and differentiation in a mouse embryonic stem cell line, MG1.19 cells

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, while Egam1c transfectants formed compacted colonies with increased E-CADHERIN expression. In Egam1c transfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genes Fgf5 (epiblast), T (mesoderm), Gata6 (primitive endoderm), and Cdx2 (trophectoderm) in -LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, including Tpbpa (spongiotrophoblast) and Plat (parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.     

牛磺酸对HepG2细胞胆固醇水平的影响

目的：探讨牛磺酸对人肝癌细胞HepG2总胆固醇（total cholesterol,TC）、游离胆固醇（free cholesterol,FC）、胆固醇酯（cholesterol ester,EC）水平及对胆固醇代谢限速酶--胆固醇7α-羟化酶（cholesterol7α-hydroxylase,CYP7A1）蛋白表达的影响。方法：在培养液中添加终浓度为0.1、1、10、20 mmol/L的牛磺酸,分别在培养24、48 h后测定细胞内TC、FC及EC水平;在培养液中添加终浓度为20 mmol/L的牛磺酸,分别培养12、24、48、72 h后检测CYP7A1蛋白表达水平。结果：1、10、20 mmol/L的牛磺酸均可使HepG2细胞内TC、FC水平明显下降,且与牛磺酸浓度呈正比,但EC水平在牛磺酸浓度达1 mmol/L后即不随牛磺酸浓度的增加而进一步降低;培养48 h后HepG2细胞TC、FC水平的降低幅度大于24 h时;20 mmol/L牛磺酸使FC含量减少的作用强于对EC减少的作用;培养24 h后HepG2细胞中CYP7A1蛋白表达最强。结论：牛磺酸可增强CYP7A1蛋白表达,促进HepG2细胞内的胆固醇代谢,胆固醇降低程度与牛磺酸浓度及作用时间相关,作用48 h的效果明显优于24 h,且牛磺酸主要是降低了细胞内的FC水平。     

Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.     

The effect of endotoxin on functional parameters of mammary CID-9 cells

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.     

Effects of epi-gallocatechin gallate on PC-3 cell cytoplasmic membrane in the presence of Cu

Catechins, which exist in green tea, are excellent antioxidants and metals enhance their pro-oxidative character. In this study, we investigated damage to the cell cytoplasmic membrane of the androgen-insensitive prostate cancer cell PC-3 from rearrangement of EGCG (epigallocatechin gallate), the main bioactive component of catechins. In a cell culture system containing Cu²⁺, the generation of free radicals from EGCG rearrangement was measured. Electron microscopy, flow cytometry, gel electrophoresis, and ESR measurements showed that the cytoplasmic membrane of PC-3 cells was disrupted in this culture system and that the death of most cells followed within a few minutes. Hydroxyl radicals, detected in the ESR experiment using the spin trapping method, may cause damage to the cytoplasmic membrane. Analysis of F-12 medium after 6 h, by HPLC, showed small amounts of EGCG when PC-3 cells were absent, and no detectable levels when PC-3 cells were present. In addition to the concentration and order in which EGCG and Cu²⁺ are added to the culture medium, oxides, polymers, or some compounds integrated with the cytoplasmic membrane of the PC-3 cells themselves may have a key role in damage to the PC-3 cell cytoplasmic membrane.     

Spermatozoa stimulate prostaglandin synthesis and secretion in bovine oviductal epithelial cells

The dynamic action of oviductal secretory compounds on spermatozoa function is well documented. In contrast, the effect of spermatozoa on oviductal function remains poorly characterized. Previously, our lab and others have shown that prostaglandin (PG), together with other vasoactive peptides, plays major roles in oviductal contractions and sperm transport. We therefore examined the effect of spermatozoa on the production of PG by cow oviductal epithelial cells (OEC). A bovine spermatozoa-OEC co-culture system was utilized for this purpose. OECs in the second passage were co-cultured for 0, 1, 3, 6, 12, and 24 h with six doses of motile, killed, or truncated spermatozoa heads (control; without spermatozoa, 10(2)-10(6) spermatozoa/ml medium). The levels of PGE(2) and PGF(2alpha) in the medium were measured using enzyme immunoassays. Messenger RNA expression of cyclooxygenase-2, PGF synthase (PGFS), and PGE synthase (PGES) was investigated using real-time RT-PCR. The results indicated that motile spermatozoa increased the secretion of PG by OEC as well as cellular expression of mRNA for cyclooxygenase, PGES, and PGFS in a dose- and time-dependent manner. A maximum three- to fivefold increased secretion of PG was observed with a dose of 10(5) spermatozoa/ml after a 12-h co-incubation. Neither killed spermatozoa nor truncated spermatozoa heads stimulated oviductal biosynthesis and secretion of PG at any dose or time point observed. The results provide the first evidence that live spermatozoa in the oviduct up-regulate the local PG system, and thereby, enhance oviductal contractions. Thus, spermatozoa may bear a role in accelerating their own transport into the fertilization site.     

Comparison of mRNA for IGFs and their binding proteins in the oviduct during the peri-oestrous period between dairy heifers and lactating cows

The oviduct provides the environment to support gamete maturation, fertilisation and early embryo development. As there is a high incidence of early embryonic death in lactating dairy cows, this study compared expression of IGF family members in the oviduct between lactating Holstein-Friesian dairy cows (n=16, 81±2.4 days in milk) and nulliparous heifers (n=16, age 1.6±0.07 years) at three stages of the oestrous cycle: A) newly selected dominant follicle in the luteal phase, B) follicular phase before the LH surge and C) pre-ovulatory phase after the LH surge. Expression of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGFBP6 mRNA was determined in the ampulla of the oviduct. Oviduct side (ipsilateral or contralateral) with respect to the dominant follicle did not affect gene expression. Expression of IGF1 and all three IGFBPs increased significantly between the luteal and the pre-ovulatory phases, with no further significant alteration post-LH surge. Concentrations of circulating IGF1 were higher in heifers than in cows, as was the mRNA expression of IGF1, IGFBP3 and IGFBP6. The pre-LH surge rise in IGFBP2 mRNA was only observed in heifers. IGF2 expression was not influenced by either age or stage of cycle. These three IGFBPs are generally considered to inhibit IGF action. These results indicate tight regulation of IGF bioavailability in the oviductal environment around oestrus, with pronounced differences between cows and heifers, which are likely to influence early embryonic development. Further studies are required to assess the implications for embryo survival.