Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

Relationships between Hha1 Calpastatin Gene Polymorphism, Growth Performance, and Meat Characteristics of Awassi Sheep.

Advances in molecular genetics have allowed the identification of genes that can enhance livestock production. The aim of this study was to investigate possible relationships between the calpastatin (CAST) Hha1 gene polymorphisms and growth performance, carcass characteristics, and meat quality in Awassi sheep. A total of 87 blood samples were collected from two-week-old Awassi ram lambs. The amplification of the CAST Hha1 gene yielded a fragment of 622 bp. Three CAST genotypes were found in Awassi sheep: MM for two fragments (385 bp and 281 bp), MN for three fragments (622 bp, 385 bp, and 281 bp), and NN for only one fragment (622 bp). The M and N allele frequencies of the CAST Hha1 genotypes were 0.765 and 0.235, respectively, while the genotypic frequencies of MM, MN, and NN were 0.586, 0.356, and 0.057, respectively. Based on CAST Hha1 gene polymorphisms, three groups of lambs (MM: n = 8; MN: n = 6; and NN: n = 3 genotypes) were subjected to a fattening period of 70 days to investigate growth performance and meat characteristics. Only the final body weight and longissimus muscle width were significantly different between the three genotypes, while no significant differences were detected in any other carcass characteristics and meat quality parameters. In this study, new variants were observed in CAST using the Hha1 restriction site, potentially assisting in Awassi sheep breeding and selection programs to improve final body weight and longissimus muscle width.

Updates on the Effect of Mycotoxins on Male Reproductive Efficiency in Mammals.

Mycotoxins are ubiquitous and unavoidable harmful fungal products with the ability to cause disease in both animals and humans, and are found in almost all types of foods, with a greater prevalence in hot humid environments. These mycotoxins vary greatly in structure and biochemical effects; therefore, by better understanding the toxicological and pathological aspects of mycotoxins, we can be better equipped to fight the diseases, as well as the biological and economic devastations, they induce. Multiple studies point to the association between a recent increase in male infertility and the increased occurrence of these mycotoxins in the environment. Furthermore, understanding how mycotoxins may induce an accumulation of epimutations during parental lifetimes can shed light on their implications with respect to fertility and reproductive efficiency. By acknowledging the diversity of mycotoxin molecular function and mode of action, this review aims to address the current limited knowledge on the effects of these chemicals on spermatogenesis and the various endocrine and epigenetics patterns associated with their disruptions.

Growth Performance and Meat Characteristics of the First Awassi-Rambouillet Callipyge Backcross.

The Awassi breed is desirable due to its resilient traits, but shows poor lean meat content on the carcass; the callipyge mutation may enhance growth and meat characteristics. The first backcross of callipyge Awassi-Rambouillet (CRAW) lambs was generated by mating Awassi ewes with heterozygous F1 (50 % Awassi and 50 % Rambouillet) rams for the callipyge mutation. A fattening trial with carriers of CLPG (CRAW), non-carriers (RAW), and Awassi (AW) ram lambs was conducted for 94 days and growth and meat characteristic parameters were recorded. Final body weight, average daily gain (ADG), feed conversion ratio, and dry matter intake, but not initial body weight, were greater in CRAW and RAW lambs as compared to AW lambs; hot and cold carcass, dressing percentage, and shoulder, rack, and loin weights were greater in CRAW vs. AW but not RAW lambs. Leg cuts were heavier in CRAW vs. both RAW and AW lambs; intermuscular and subcutaneous fat percentages were greater in CRAW as compared to AW but not RAW lambs. Non-carcass components showed kidney fat, kidney, liver and testes weights higher in CRAW lambs as compared to AW lambs, but these parameters were similar to those of RAW lambs. There were no differences in fat tail weight. Further, CRAW lambs had higher shear force and water holding capacity than RAW and AW lambs, while cooking loss was the lowest in CRAW lambs. In conclusion, the callipyge mutation with 25% Rambouillet genes can provide efficient improvements in growth and meat characteristics, with the exception of tenderness in Awassi sheep.

The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems.

Hedgehog signaling is involved in regulation of ovarian function in Drosophila, but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or theca (TC) cells of small (1-5 mm) antral follicles by in situ hybridization and of larger (5-17 mm) antral follicles by real-time RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiol > progesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1-treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors.

Effect of resistin on granulosa and theca cell function in cattle.

Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1-5mm) and/or large (8-22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.

The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells.

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased 4-24 h after hCG versus 0-48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

Role of adiponectin in regulating ovarian theca and granulosa cell function.

Adiponectin is an adipokine that has been implicated in insulin resistance, a condition associated with polycystic ovarian syndrome in humans, but whether adiponectin can directly affect ovarian theca or granulosa cell function is unknown. Therefore, to determine the effects of adiponectin on proliferation, steroidogenesis and gene expression of large-follicle theca and granulosa cells, experiments were conducted using bovine ovarian cell cultures. RT-PCR was used to elucidate the effects of adiponectin on gene expression of CYP11A1 and LH receptor (LHR) in large-follicle theca and granulosa cells, as well as expression of CYP17A1 in theca cells and CYP19A1 in granulosa cells. Adiponectin decreased (P<0.05) insulin-induced progesterone and androstenedione production as well as attenuated IGF-I-induced LHR, CYP11A1, and CYP17A1 gene expression in theca cells. In contrast, adiponectin decreased (P<0.05) LHR mRNA abundance in granulosa cells but did not affect steroidogenic enzyme gene expression in granulosa cells. Adiponectin had no effect (P>0.10) on proliferation of large-follicle theca cells. RT-PCR also revealed that abundance of mRNA for the adiponectin receptor (ADIPOR2) was greater (P<0.05) in large-follicle than in small-follicle theca cells and did not significantly differ between small- and large-follicle granulosa cells. In cultured theca cells, LH increased (P<0.05) and IGF-I decreased (P<0.05) ADIPOR2 mRNA abundance. These results indicate that the inhibitory effects of adiponectin on steroidogenesis are primarily localized to theca cells and that the response of theca cells to adiponectin (i.e., ADIPOR2) may be regulated by LH and IGF-I.

High-fat diets promote insulin resistance through cytokine gene expression in growing female rats.

To determine if tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). IL-6 gene expression is influenced by amount and source of dietary fat, 30 weanling female rats were randomly assigned to a moderate-fat soybean oil (MFS; 22% of total energy fed as fat), high-fat (HF) soybean oil (HFS; 39% of total energy fed as fat), or HF tallow (HFT; 39% of total energy fed as fat) diet treatments. Oral glucose tolerance tests (OGTT) were conducted serially over 10 weeks of treatment. HFT and HFS rats gained more weight and had greater body fat than the MFS rats fed similar amounts of energy. Both groups of HF-fed rats had greater (P<.05) insulin resistance (homeostasis model assessment) than MFS-fed rats. TNF-alpha mRNA abundance quantified by real-time reverse transcriptase-polymerase chain reaction was greater (P<.05) in liver and lower (P<.05) in adipose tissue in HFT compared to HFS and MFS rats. There were positive correlations (P<.05) between hepatic TNF-alpha mRNA and insulin resistance, and negative correlations between insulin sensitivity and hepatic TNF-alpha mRNA and hepatic IL-6 mRNA. During Week 3 and Week 6 OGTTs, hyperinsulinemic responses were observed in the HFT group, after which, on Week 9, insulin secretion was diminished in response to the OGTT, suggesting impaired pancreatic insulin secretion. HFS rats exhibited insulin resistance on Week 9 OGTT. In summary, an HFT diet fed to growing female rats caused insulin resistance associated with increased hepatic TNF-alpha mRNA leading to pancreatic insufficiency. Early-onset insulin resistance related to the inflammatory process in obesity is influenced by the amount and type of fat in the diet.

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9.

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.

Effects of Fusarium mycotoxins on steroid production by porcine granulosa cells.

Fusarium mycotoxins, such as trichothecenes and zearalenone, are common grain and foodstuffs contaminants. Some of these like deoxynivalenol (DON) can negatively impact pregnancy success in swine, but evidence for direct ovarian effects of DON, zearalenone, and its major metabolite, alpha-zearalenol (ZEA) is meager. To evaluate the effects of two mycotoxins, DON and ZEA on porcine granulosa cell(s) (GC) proliferation, steroidogenesis and gene expression, pig GC from small follicles (1-5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing control (no mycotoxins) or mycotoxins (at various doses/combinations). Both DON and ZEA had biphasic effects on IGF-I-induced estradiol production, increasing estradiol production at smaller doses and inhibiting at larger doses. ZEA at 3,000 ng/mL (9.37 microM) increased IGF-I-induced progesterone production and at 30 ng/mL (0.0937 microM) and 300 ng/mL (0.937 microM) were without effect, but these doses of ZEA increased FSH-induced progesterone production. ZEA at 3,000 ng/mL inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. DON inhibited progesterone production at 100 ng/mL (0.337 microM) and 1,000 ng/mL (3.37 microM) but at 10 ng/mL (0.0337 microM) was without effect. DON at 1,000 ng/mL (but not at 10 ng/mL) completely inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. The concomitant treatment of ZEA had little effect on the dose response to DON. DON increased IGF-I-induced cell numbers at 10 and 100 ng/mL and inhibited cell numbers at 1,000 ng/mL, whereas ZEA had no effect on GC numbers. Only a combined treatment of DON and ZEA increased serum-induced cell proliferation. In conclusion, mycotoxins have direct dose-dependent effects on GC proliferation, steroidogenesis and gene expression. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine.

Real-time RT-PCR quantification of pregnancy-associated plasma protein-A mRNA abundance in bovine granulosa and theca cells: effects of hormones in vitro.

Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins -2, -4 and -5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1-5 mm) and large (LG) (8-22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3-500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1-10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P>0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P>0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P<0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle.

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.