Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.     

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.     

胰岛素样生长因子对胃肠道影响的研究与展望

综述了有关IGFs基础研究方面的进展，特别是对小鼠、仔猪及新生儿胃肠道及其黏膜系统之间的关系等方面的进展。这为合理有效地利用IGFs重要资源（特别是牛初乳资源）提供研究的科学依据和途径。     

Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles

Follicles of 4-8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 mug/ml) at 39 degrees C in a humidified atmosphere of 45% O2, 5% CO2 and 50% N2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 +/- 8.53 vs 124.6 +/- 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.     

Gene expression for LH receptor, 17 alpha-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17 alpha-hydroxylase (17 alpha OH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17 alpha OH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17 alpha OH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR. In summary, our study suggests that in spite of the presence of 17 alpha OH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.     

Changes in fibroblast growth factor 2 and its receptors in bovine follicles before and after GnRH application and after ovulation

The aim of this study was to evaluate the expression pattern of fibroblast growth factor 2 (FGF2), its receptor variants (FGFR1IIIc, FGFR2IIIc) and nucleolin in time-defined follicle classes before and after GnRH application and after ovulation in the cow. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 4, 10, 20 and 25 h (follicles) and 60 h (new CL) relative to injection of GnRH to induce an LH surge (n = 5 animals per group). The expressions of FGF2 and FGFR1IIIc mRNA were significantly up-regulated only in the follicle group 4 h after GnRH (during the LH surge) with a significant down-regulation immediately afterwards. Western blot analyses showed two protein bands with at 22 and 18 kDa with apparent up-regulation beginning with the LH surge (4 h) and maximum levels 20 h after GnRH. FGF2 protein in follicles collected at 0 h (before LH surge) was localised in theca tissue (endothelial and pericytes of blood vessels) but not in granulosa cells (GCs). The FGF2 staining (by immunohistochemistry) pattern changed dramatically after the LH surge for a short period (about 2 days) and FGF2 protein was localised dominantly in the nucleus of many GCs, while most capillary endothelial cells were FGF2 immunonegative. In conclusion, the novel observation of FGF2 up-regulation and the distinct change in FGF2 localisation from theca (cytoplasm of endothelial cells) to the nucleus of GCs after the LH surge may be important for survival of GCs or for the transition of the GCs to luteal cells.     

Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.     

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.     

Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.     

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.     

Changes in insulin-like growth factor binding protein (IGFBP) isoforms during bovine follicular development

UThe insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and so regulate their access to the type 1 and 2 IGF receptors. This is the principal mechanism involved in regulating IGF bioavailability during folliculogenesis. IGFBPs undergo a number of post-translational modifications, including proteolytic cleavage, phosphorylation and glycosylation, which can regulate the affinity of IGFBPs for IGFs. However, the post-translational changes to IGFBPs that occur during folliculogenesis have not been fully characterized. The charge and size variants of the IGFBPs in bovine follicular fluid were examined by two-dimensional non-reducing SDS-PAGE followed by non-isotopic western ligand blot analysis, and immunoblot analysis during follicular development. The results demonstrate the presence of at least 51 IGFBP isoforms corresponding to IGFBP-1 to -6 in bovine follicular fluid from subordinate follicles, many of which were phosphorylated. The total number of IGFBPs was reduced in dominant follicles, whereas no gross changes in isoforms were observed during follicular development. These results demonstrate the high degree of conservation of IGFBP post-translational modifications between species, and from the in vitro dephosphorylation of these proteins it is hypothesized that these modifications may result in changes to IGF binding or susceptibility to proteolytic cleavage.     

Stimulatory effects of fasting on vascular endothelial growth factor (VEGF) production by growing pig ovarian follicles

The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.     

Effect of negative energy balance on the insulin-like growth factor system in pre-recruitment ovarian follicles of post partum dairy cows

Post partum negative energy balance (NEB) in dairy cattle is associated with a delayed return to ovarian cyclicity and reduced fertility. This study compared the IGF system of pre-recruitment ovarian follicles between cows in mild (n = 6) or severe (n = 6) NEB during early lactation. Ovaries were collected in the second week post partum, when circulating concentrations of IGF-I and glucose were lower (P < 0.01) in severe NEB cows. mRNA expression for IGF-II, type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1 to IGFBP-6 was determined by in situ hybridisation in individual follicles using radiolabelled oligonucleotide probes. Follicles were classified as very small (1-2.5 mm) or small (2.5-5 mm) and healthy or atretic. Relative mRNA concentrations were measured as optical density (OD) units using image analysis. Thecal IGF-II mRNA expression was highest in very small, healthy follicles (P < 0.05). Granulosa cell IGFBP-2 was the only component to change with EB status, with higher mRNA expression in mild compared with severe NEB cows (P < 0.05). IGFBP-1 and IGFBP-3 mRNA expression were undetectable. IGF-1R, IGFBP-4 and IGFBP-5 mRNA expression were not significantly altered by follicle size or health, but IGFBP-5 tended to increase in atretic follicles. The pattern of IGFBP-6 mRNA expression in theca paralleled that of IGF-II mRNA, with higher (P < 0.05) levels in healthy, very small follicles. In conclusion, the reduced expression of IGFBP-2 mRNA in severe NEB cows may alter the bioavailability of circulating IGF-I and locally produced IGF-II to modulate the pre-recruitment stages of follicles required to maintain normal post partum ovarian cyclicity.     

Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents

Folliculogenesis is a complex process regulated by various paracrine and autocrine factors. In vitro growth systems of primordial and preantral follicles have been developed for future use of immature oocytes, as sources of fertilizable oocytes and for studying follicular growth and oocyte maturation mechanisms. Rodents were often chosen for in vitro follicular culture research and a lot of factors implicated in folliculogenesis have been identified using this model. To date, the mouse is the only species in which the whole process of follicular growth, oocyte maturation, fertilization and embryo transfer into recipient females was successfully performed. However, the efficiency of in vitro culture systems must still be considerably improved. Within the follicle, numerous events affect cell proliferation and the acquisition of oocyte developmental competency in vitro, including interactions between the follicular cells and the oocyte, and the composition of the culture medium. Effects of the acting factors depend on the stage of follicle development, the culture system used and the species. This paper reviews the action of endocrine, paracrine factors and other components of culture medium on in vitro growth of preantral follicles in rodents.     

Seasonal effects on the response of ovarian follicles to IGF1 in mares

The response of follicles to IGF1 was compared between the transition into the ovulatory season (transitional period) and the ovulatory season (ovulatory period) in eight mares using a cross-over experimental design within periods. Granulosa cells were collected from follicles 15-24 or 25-34 mm and expression of IGF1R, IGF2R, FSHR, LHCGR and PAPPA was determined by qPCR. In addition, 10 mg IGF1 or vehicle were injected into the largest follicle (transitional period) or the second largest follicle (ovulatory period) of a follicular wave before the beginning of diameter deviation between the two largest follicles (mean diameters at injection 19.2 and 20.0 mm during transitional and ovulatory periods respectively). Follicular fluid was collected 24 h after injection for determination of free IGF1, IGFBP, inhibin A and oestradiol levels. Granulosa cells from follicles 25-34 mm, but not follicles 15-24 mm, expressed higher levels of IGF1R (P=0.01), FSHR (P<0.007) and LHCGR (P=0.09) during the ovulatory period than during the transitional period, whereas IGF2R expression was higher in transitional than ovulatory follicles (P=0.06). Follicular IGFBP2 levels were not different (P>0.1) between periods and treatments, whereas IGFBP5 levels were higher (P<0.05) during the ovulatory period. Finally, IGF1 injection before the beginning of deviation induced an approximately twofold increase (P=0.01) in follicular inhibin A levels during each period and did not affect oestradiol (P>0.1). These results suggest that, as during ovulatory waves, equine follicles during transitional waves are responsive to IGF1 before the beginning of deviation and that, therefore, inadequate IGF1 responsiveness before deviation may not underlie the deficient development of dominant follicles during transition.     

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes

Cloning mammalians by somatic cell nuclear transfer (SCNT) remains inefficient. A majority of clones produced by SCNT fail to develop properly and of those which do survive, some exhibit early aging, premature death, tumors, and other pathologies associated with aneuploidy. Alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types. It remains to be determined how centrosomes are remodeled in cloned bovine embryos. We show that abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos following SCNT. Moreover, centrosome abnormalities and failed 'pronuclear' migration which manifested during the first cell cycle coincided with errors in spindle morphogenesis, chromosome alignment, and cytokinesis. By contrast, nuclear mitotic apparatus protein (NuMA) exhibited normal expression patterns at metaphase spindle poles and in 'pronucleus' during interphase. The defects in centrosome remodeling and 'pronuclear' migration could lead to chromosome instability and developmental failures associated with embryo production by SCNT. Addressing these fundamental problems may enhance production of normal clones.     

In vitro development of pig preantral follicles cultured in a serum-free medium and the effect of angiotensin II

A novel culture system is reported in which pig preantral follicles (< 300 microm in diameter) with an intact thecal cell layer were isolated and cultured in a serum-free medium for up to 30 days. The medium supported follicle culture after isolation, while maintaining both somatic cell and oocyte viability. Follicles were cultured in groups (n = 3 per group) on collagen-coated wells for 16 days, during which they retained a three-dimensional structure, maintained oocyte viability and increased in diameter and number of somatic cells. Follicle culture for 30 days resulted in a further increase in number of cells, oocyte viability was maintained, and a significant increase in follicle diameter was observed (P < 0.001), with 29% of follicles forming an antrum. Follicles synthesized measurable quantities of progesterone (168 pg per 100 microl per 48 h; no significant increase with time) and increasing quantities of oestradiol (136 pg per 100 microl per 48 h; P < 0.001 with time). Further supplementation of the medium with 100 micromol testosterone l(-1) at day 28 resulted in a significant increase in oestradiol secretion by both antral (P < 0.01) and preantral follicles (P < 0.05). Culture over 30 days in medium with 10(-10) mol angiotensin II l(-1) and further supplementation at day 28 with 100 micromol testosterone l-1 also increased oestradiol synthesis (P < 0.001). These results show that viable preantral follicles may be cultured for extended periods, and indicate that the possible role of angiotensin II in folliculogenesis and steroidogenesis in early development of pig follicles requires further investigation.     

Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro

Hepatocyte growth factor (HGF) regulates granulosa cell (GC) steroidogenesis and suppresses apoptosis in non-ovarian cells. The hypothesis was thus developed that intraovarian HGF supports folliculogenesis by mediating steroidogenesis and suppressing apoptosis. To investigate the latter, the anti-apoptotic actions of HGF were tested in GCs and follicles isolated from immature rats. Results showed that HGF suppressed apoptosis in GC and follicle cultures as visualized using apoptosis indicator dye, YO-PRO-1. Immunohistochemistry was used to investigate the distribution of HGF, c-met, and HGF activator (HGFA) protein during folliculogenesis in equine chorionic gonadotropin (eCG)-primed rats. Immunoreactive HGF content was the greatest in GCs within preantral follicles. Following eCG, large antral follicles showed elevated HGF staining in theca and interstitial cells when compared with GCs. Intense c-met staining was observed in GCs within non-primed small preantral follicles; following eCG, the level of c-met was diminished in GCs, but increased within theca and interstitial cells. Theca, interstitium, and GCs in non-primed and primed ovaries contained HGFA. Following eCG, HGFA was more apparent in theca cells and the interstitium when compared to that in GCs within large antral follicles. The presence of HGF, c-met, and HGFA in preantral follicles would potentially enable the anti-apoptotic effects of HGF that were observed in vitro to occur in vivo. Advanced folliculogenesis led to a change in the cellular distribution of the HGF, c-met, and HGFA, suggesting that the ovarian HGF system is hormonally regulated in vivo.     

Percentages of protein and nonprotein nitrogen with varying fat and somatic cells in bovine milk

Over 15 mo, 28,390 individual milk samples from 3,600 cows in 62 Quebec Holstein herds were analyzed for crude protein, true protein, nonprotein nitrogen, fat, and somatic cell counts. Unadjusted means with standard errors were 3.51% +/- .002, 3.31% +/- .002, 31.70 mg/100 ml +/- .12, 3.67% +/- .004, and 297,230 cells/ml +/- 4002. Nonprotein nitrogen as a percentage of total nitrogen was 5.57% +/- .02. Least squares analyses showed significant effects of herd, age of cow, month of test, stage of lactation, somatic cell count, and fat percentage on contents of crude protein, true protein, and ratio of nonprotein nitrogen to total nitrogen. Highly variable nonprotein nitrogen fraction content during different months of the year and various stages of lactation is responsible for changes of crude protein content whereas changes of crude protein for different ages of cows, fat, and somatic cells in milk are due to changes of true protein content. Automatic infrared instrument calibrated against crude protein standards can be used satisfactorily to measure crude protein in milk if variations are due to age of cow, fat, and somatic cell counts. However, the instrument becomes inaccurate for measuring large variation of crude protein caused by variability of nonprotein nitrogen due to season and stage of lactation.     

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.