Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo

To evaluate the regulation of ovarian insulin-like growth factor-I (IGF-I) during follicular growth in vivo, we measured the concentration of this peptide in follicular fluid (FFL) of immature gilts during the induction of follicular development by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). FFL concentrations of immunoreactive (i) IGF-I were compared with those of intrafollicular steroids and with concentrations of iIGF-I, estradiol (E2), and porcine growth hormone (GH) in serum. PMSG, administered at Time 0, induced a significant (p less than 0.01), time-dependent increase in intrafollicular iIGF-I that peaked 72 h after administration of the hormone, before the administration of hCG. During the first 72 h, the changes in ovarian iIGF-I paralleled those for progesterone and E2. After the administration of hCG at 72 h, FFL levels of E2 fell, those of iIGF-I remained constant, and progesterone rose. Serum E2 concentrations paralleled those in FFL. Since serum GH and IGF-I levels rise during spontaneous puberty in some species, these levels were also monitored. However, a significant treatment effect on serum GH and iIGF-I was not demonstrated. In summary, ovarian concentrations of iIGF-I are increased by gonadotropic hormones in vivo. The absence of concomitant changes in circulating levels of iIGF-I and GH suggests that the gonadotropin effects are exerted directly on the ovary. These results, together with more abundant data regarding secretion and action of IGF-I in cultured granulosa cells, suggest that IGF-I may function in an autocrine or paracrine fashion to amplify the actions of gonadotropins at an ovarian level.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.     

Identification and NH2-terminal amino acid sequence of three insulin-like growth factor-binding proteins in porcine serum.

Three distinct species of IGFBP in porcine serum were identified by NH2-terminal amino acid sequence analysis. The IGFBPs identified include pIGFBP-2 (34 kDa), three isoforms of pIGFBP-3 (43, 40 and 30 kDa) and two isoforms of pIGFBP-4 (30 and 26 kDa). The three isoforms of pIGFBP-3 were found to have a common NH2-terminal amino acid sequence, as were the two isoforms of pIGFBP-4. These results indicate that porcine serum contains a truncated form of IGFBP-3 and two forms of pIGFBP-4, similar to those previously isolated from human and rat serum. Furthermore, the presence of a truncated form(s) of the GH-dependent IGFBP-3 in porcine serum suggests that elucidating its origin and function may be important in understanding how IGFBPs affect the somatogenic actions of GH.     

Identification and hormonal control of reproductive-tract-specific antigens present in rabbit oviductal fluid

Utilizing the intra-abdominal flask technique to collect oviductal fluid, the presence of two or possibly three reproductive-tract-specific antigens have been observed in rabbit oviductal fluid. Two of these antigens may be accounted for by the two forms of uteroglobin. The other antigen has a molecular weight greater than 200,000 daltons and its concentration in oviductal fluid is under hormonal control. During pseudopregnancy (PSP), when progesterone concentrations are high, or upon progesterone administration, the concentration of this high molecular weight antigen doubles in oviductal fluid. This correlates well with the previously observed increase in release of secretory products from the oviductal epithelia.     

Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency

We report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory [Ikeda, T., & Sirbasku, D.A. (1984) J. Biol. Chem. 259, 4049-4064]. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 200 micrograms/mL bovine serum albumin, and 10 micrograms/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The isolation of UDGF began with acetic acid extraction followed by sulfopropyl-Sephadex chromatography, Bio-Gel P-10 molecular sieve fractionation, and a series of reverse-phase high-pressure liquid chromatography separations. Purifications [[(1.0-8.5) X 10(6)]-fold] of three mitogens (5-20 ng each) were achieved. The mitogens were shown by protein microsequencing to be DES 1----3 to DES 1----6 forms of insulin-like growth factor I (truncated IGF-I). An Mr estimated by 125I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1----3(4) N alpha truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. The ED50 values of DES 1----3 IGF-I and recombinant IGF-I for MCF-7 cell growth were 0.8-6.0 and 30-150 pg/mL, respectively. With Balb/c 3T3 mouse fibroblasts, the ED50 of DES 1----3 IGF-I was 100 times lower than that of IGF-I. We conclude that the major acid-stable low-Mr mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.     

Development of porcine embryos from one- and two-cell stages to blastocysts in culture medium supplemented with porcine oviductal fluid

Oviductal fluid (OVF) was harvested chronically from 5 sows beginning on Day 1 of the estrous cycle (Day 0 of estrous cycle = day of detected estrus) and used for embryo culture (Day 3 OVF only). Two experiments were conducted to investigate in vitro development of 1-cell and 2-cell porcine embryos in a modified Kreb's Ringer bicarbonate medium (culture medium, CM), early luteal phase OVF or CM supplemented with OVF (CM-OVF, 25% OVF v/v in CM) with or without transfer to fresh CM. In Experiment 1, 1-cell and 2-cell embryos were harvested from sows (n = 7) approximately 44 h after detected estrus. In Experiment 2, 1-cell embryos were collected from 5 sows treated with altrenogest and gonadotropins, approximately 50 h after injection of human chorionic gonadotropin. The volume of OVF (ml) declined progressively throughout the 4 days of collection (24 h, 8.44 +/- 0.28; 48 h, 6.88 +/- 1.78; 72 h, 4.96 +/- 0.35; 96 h, 4.64 +/- 0.25 after onset of estrus; p less than .01). In both experiments, development to blastocyst stage was lowest among embryos cultured in OVF and highest among those cultured in CM-OVF (Experiment 1: CM, 27.3; OVF, 10; CM-OVF, 63.6; Experiment 2: CM, 26.7; OVF, 0; CM-OVF, 82.4; % blastocyst formation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

Caloric restriction and insulin-like growth factors in aging and cancer.

This review examines the influence of a reduction in caloric intake on cancer and longevity. Data indicating that rodents subjected to caloric restriction display lower tumor incidence, reduced susceptibility to carcinogens and extended lifespan is analyzed. The potential role of the growth hormone/insulin-like growth factor type 1 (IGF-I) axis in this effect is discussed as is the evidence that a reduction in growth hormone and/or IGF-I leads to a reduction in spontaneous tumors and susceptibility to carcinogens.     

Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.     

The role of insulin-like growth factors in small intestinal cell growth and development.

IGF-I and IGF-II receptors are expressed in the small intestine of mammalian species, as are the genes to synthesize both peptides. IGF binding proteins are also expressed in the intestine. IGF-I and IGF-II mRNA are highest in fetal and newborn tissues and decrease with age. IGF-I mRNA is present in the adult small intestine, and is associated with the submucosal regions and crypt cells. IGF-I and IGF-II receptor binding to the small intestine is higher in newborn animals and decreases with age. Both receptors are more concentrated in the crypt than villus regions, but IGF-II binding is higher than IGF-I in all regions. IGF-I receptors are associated with the submucosal region of the small intestine, whereas IGF-II receptors are more abundant in the mucosal cells. Administration of IGF-I either orally or by osmotic pump generally has no affect on small intestinal weight or length, but does increase mucosal cellularity. LR3-IGF-I administration by osmotic pump affects the small intestine similarly to IGF-I, although with a higher potency. In the few studies in which IGF-II was administered, increased gut mass was observed in adult rats, but not newborn rats or pigs. Significant effects on mucosal expression of disaccharidases was achieved with either oral or subcutaneous IGF-I or oral IGF-II. Administration of IGF in models of intestinal hypertrophy and atrophy are also reviewed.     

Identification of a conserved microsatellite site in the porcine and bovine insulin-like growth factor-I gene 5' flank

Examination of published rat and human sequences for the insulin-like growth factor-I (IGF-I) gene indicated the presence of CA dinucleotide repeats in corresponding segments of each. Presence of similar microsatellite sequences in the porcine and bovine IGF-I genes was hypothesized. A 1200-bp segment upstream of the porcine and bovine IGF-I genes was amplified using the polymerase chain reaction (PCR) with primers developed from a consensus of human, rat and bovine sequences. Both porcine and bovine PCR products contained similar microsatellite sequences. Amplified pIGF-I DNA was cloned and sequenced, and an additional primer was developed specifically for microsatellite marker detection. Six allelic variants of 124, 130, 132, 134, 136 or 138 bp were observed in pigs with differing frequencies between breeds (P < 0.01). The same primers were used to amplify the corresponding bovine microsatellite. Three alleles of 126, 128 and 130 bp were observed in a genetically diverse cattle population with estimated frequencies of 0.06, 0.68 and 0.26, respectively. Results of this study indicate sequence information from the human and laboratory species can be used to facilitate genetic marker development in livestock species.     

Nutrition, insulin, insulin-like growth factors and cancer.

The incidence of colon, pancreatic, and kidney cancers, as well as aggressive prostate cancer in men, and breast and endometrial cancer in women is invariably high in Western countries. Nutritional and related factors have been typically implicated. This review presents a model integrating nutrition, insulin and IGF-1 physiology ("bioactive" IGF-1), and carcinogenesis based on the following: (1) insulin and the IGF-1 axis function in an integrated fashion to promote cell growth and survival; (2) chronic exposure to these growth properties enhances carcinogenesis; (3) factors that influence bioactive IGF-1 will affect cancer risk. The model presented here summarizes the data that chronic exposure to high levels of insulin and IGF-1 may mediate many of the risk factors for some cancers that are high in Western populations. This hypothesis may help explain some of the epidemiologic patterns observed for these cancers, both from a cross-national perspective and within populations. Of particular importance is that some of relevant factors are modifiable through nutritional and lifestyle interventions. Out of a variety of perspectives presented, nutritional manipulation through the insulin pathway may be more feasible than attempting to influence total IGF-1 concentrations, which are determined largely by growth hormone. Further study is required to test these conclusions.     

Structural characterization of a follicle-stimulating hormone action inhibitor in porcine ovarian follicular fluid. Its identification as the insulin-like growth factor-binding protein.

Recently, an inhibitory polypeptide that could block the follicle-stimulating hormone-induced estradiol and progesterone production in rat ovary granulosa cells has been isolated from porcine ovarian follicular fluid. Amino-terminal sequence analysis of the purified inhibitor suggests that it could be the porcine congener of the 53-kDa subunit of the growth hormone-dependent insulin-like growth factor binding protein (IGF-BP3). Using amino acid sequence information derived from the purified inhibitor to construct oligonucleotide probes, we have now identified the complementary deoxyribonucleic acids (cDNAs) encoding the inhibitory polypeptide from a porcine liver and a porcine ovary library. The nucleotide and predicted amino acid sequences revealed that the cDNAs indeed encode the porcine homolog of the recently characterized human IGF-BP3. The mature polypeptide consists of 266 amino acids, which is 2 amino acids longer than the human sequence. Between the two species, there are 42 amino acid substitutions, but the 18 cysteines and the three Asn-linked glycosylation sites are totally conserved. A single mRNA species of 2.6 kilobases encoding the IGF-BP3 was detected in porcine gonadal, brain, and liver tissues by Northern analysis.     

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Dose-response study of intrafollicular injection of insulin-like growth factor-I on follicular fluid factors and follicle dominance in mares

The effect of insulin-like growth factor-I (IGF-I) on the concentrations of follicular fluid factors during follicle deviation and the development of dominance was studied in mares in two experiments. Transvaginal ultrasound guidance was used for intrafollicular injection and subsequent sequential sampling of follicular fluid. Treatment involved a single injection of IGF-I into the second-largest follicle (F2) at the expected beginning of deviation (Hour 0) based on diameter (> or =20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 microg (experiment 1) or 250, 25, or 2.5 microg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-microg doses stimulated growth, leading to ovulation of F2 in 10 of 10 and 4 of 5 mares in the two experiments, respectively, compared to 4 of 12 and 0 of 5 in saline-injected controls. These doses prevented (P < 0.05) the increase in IGF binding protein-2 and androstenedione that occurred in F2 of controls and increased (P < 0.05) the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF). The 500-microg dose stimulated higher (P < 0.05) concentrations of estradiol, but not until Hour 48, whereas the lower doses were ineffective. In experiment 2, free IGF-I concentrations in F2 at Hour 24 decreased progressively as the dose decreased so that concentrations for the 2.5-microg dose were higher (P < 0.05) than in F2 of controls and similar (not significantly different) to endogenous concentrations in F1. Correspondingly, concentrations of androstenedione in F2 at Hour 24 were lower (P < 0.05) and concentrations of activin-A, inhibin-A, and VEGF were higher (P < 0.05) after treatment of F2 with the 2.5-microg dose than in F2 of controls and were similar to concentrations in F1. Hence, a physiologic intrafollicular dose of IGF-I did not stimulate estradiol production but reduced the production of androstenedione and stimulated the production of activin-A, inhibin-A, and VEGF during follicle selection in mares.