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The Adipose Obese Gene Product, Leptin: Evidence of a Direct Inhibitory Role in Ovarian Function¹

Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078-0425

Address all correspondence and reprint requests to: Leon J. Spicer, Ph.D., Department of Animal Science, Oklahoma State University, 114 Animal Science Building, Stillwater, Oklahoma 74078-0425.

	Abstract

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Leptin, a recently-discovered hormonal product of the obese gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety and improves reproductive performance if injected into mice lacking circulating leptin. Therefore, we tested the hypothesis that leptin signals metabolic information to the reproductive system by directly affecting granulosa cell function. Doses of 10–300 ng/ml leptin had no effect (P > 0.10) on basal or insulin-induced numbers of granulosa cells cultured from small (1–5 mm) or large (8 mm) bovine follicles. Similarly, 30 and 300 ng/ml leptin had no effect (P > 0.10) on basal estradiol production. However, leptin, in a dose-dependent manner, inhibited (P < 0.05) insulin-induced progesterone and estradiol production by granulosa cells from small and large follicles. Leptin did not compete for specific ¹²⁵I-insulin binding to granulosa cells. Furthermore, specific binding of ¹²⁵I-leptin was demonstrable in granulosa cells. In conclusion, leptin, at physiological levels, can directly attenuate insulin-induced steroidogenesis of granulosa cells without affecting proliferation of this ovarian cell type. These results provide evidence to support the hypothesis that leptin can act as a metabolic signal to the reproductive system via direct action at the ovarian level.

	Introduction

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THE RECENTLY identified obese (ob) gene product, leptin, is a plasma protein hormone that parallels the amount of fat reserves (1, 2, 3, 4, 5) and is thought to regulate satiety (6, 7, 8, 9). The amino acid sequence of leptin is highly conserved (i.e. 84–97% homology) across the four species (mouse, rat, human, cattle) in which it has been characterized (10, 11, 12, 13). If genetically obese (ob/ob) mice that lack endogenous leptin are injected with exogenous leptin, they experience decreased food intake, a loss in body weight, increased ovarian weight and number of follicles, and correction of a sterility defect (14, 15). The latter observations indicate that leptin may have positive influences on the reproductive system. However, the majority of women with polycystic ovary disease are obese, insulin-resistant, and anovulatory (16, 17, 18), suggesting that leptin also may have negative impacts on the reproductive system.

Recent evidence shows that patients with noninsulin-dependent diabetes mellitus have significantly greater leptin levels than their respective control groups (1, 19). Furthermore, noninsulin-dependent diabetes mellitus patients are insulin-resistant (1), but whether leptin directly influences a tissue’s response to insulin is unknown.

Insulin has long been known to have direct effects on ovarian cell function (20, 21) and thought to be involved in the clinical manifestation of increased androgenicity in polycystic ovary patients (21, 22). Obesity also has been linked to obstetric complications (23) and breast cancer (24). Therefore, we hypothesized that leptin may directly influence insulin-stimulated ovarian follicular function, and we set out to determine the effect of leptin on insulin-induced proliferation and steroidogenesis of granulosa cells in vitro.

	Materials and Methods

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Reagents and hormones
Reagents were DMEM, Ham’s F12, insulin (bovine; 25.7 U/mg), enzymes, and FCS, all obtained from Sigma Chemical Co. (St. Louis, MO); bovine FSH (F1913, FSH activity 15 x NIH-FSH-S1 U/mg) obtained from Scripps Laboratories (San Diego, CA); ¹²⁵I-insulin (specific activity = 50–80 µCi/µg) obtained from ICN Biomedicals Inc. (Irvine, CA); and recombinant mouse leptin obtained from Pepro Tech Inc. (Rocky Hill, NJ).

Cell culture
Ovaries were obtained at a nearby commercial abattoir from beef and dairy cattle after slaughter. After transport to the laboratory on ice (<120 min), the ovaries were processed and granulosa cells isolated, as described previously (25, 26). Briefly, granulosa cells from small (1–5 mm) undifferentiated follicles and large (8 mm) moderately differentiated follicles were collected by aspiration using a needle and syringe and then washed twice in serum-free medium (25). Cells were resuspended in serum-free medium, and the number of viable cells was determined using the trypan blue exclusion method. Cell viability averaged 25 ± 4% and 29 ± 6% for small-follicle and large-follicle granulosa cells, respectively, at the time of plating.

Medium was a 1:1 (vol/vol) mixture of DMEM and Ham’s F-12 containing 0.12 mM gentamicin and 38.5 mM sodium bicarbonate. Approximately 2 x 10⁵ viable cells in 20–105 µl of medium were added to Falcon multiwell plates (no. 3047; Becton Dickinson and Co., Lincoln Park, NJ) containing 1 ml of medium. Cultures were kept at 38.5 C in a 5% CO₂ atmosphere. To obtain optimal attachment, cells were maintained in the presence of 10% FCS for the first 2 days of culture. Plating efficiency averages 24% for bovine granulosa cells under these conditions (27). After this time, cells were washed twice with 0.5 ml serum-free medium, and incubations continued in serum-free medium with or without added hormones. Medium was changed every day. For experiments evaluating the effects of hormones on steroid production, hormonal treatments were applied for 24 h (i.e. from day 2 to day 3 of culture), unless stated otherwise.

Exp 1 and Exp 2 were conducted to evaluate the dose-response effect of leptin on the action of FSH and insulin on cell proliferation and steroidogenesis of granulosa cells collected from small and large follicles, respectively. In Exp 1, granulosa cells collected from small (1–5 mm) follicles were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 24 h with 500 ng/ml testosterone and 50 ng/ml FSH in the absence or presence of 100 ng/ml insulin and leptin (0, 10, 30, 100, and 300 ng/ml). In Exp 2, granulosa cells from large (8 mm) follicles were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 24 h with 500 ng/ml testosterone and 50 ng/ml FSH in the absence or presence of 100 ng/ml insulin and leptin (0, 10, 30, 100, and 300 ng/ml), as in Exp 1. The doses of insulin and FSH were selected based on previous studies (28, 29). Concentrations of leptin in blood of lean and obese humans have been reported to be between 2–10 ng/ml and 10–100 ng/ml, respectively (1, 2, 3, 4, 5).

Exp 3 and Exp 4 were conducted to evaluate the effect of leptin, in the absence of insulin, on granulosa cell numbers and steroidogenesis of granulosa cells collected from small and large follicles, respectively. In Exp 3, granulosa cells collected from small (1–5 mm) follicles were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 24 h with 500 ng/ml testosterone in the absence or presence of 50 ng/ml FSH and leptin (0, 30, or 300 ng/ml). In Exp 4, granulosa cells from large (8 mm) follicles were cultured and treated as described for Exp 3. The doses of leptin were selected based on results from Exp 1 and Exp 2.

Exp 5 was conducted to evaluate whether the inhibitory effect of leptin on insulin-induced granulosa cell steroidogenesis was caused by leptin inhibiting insulin from binding to its receptors. Granulosa cells were cultured for 3 days in 10% FCS, and then medium was removed, cells washed twice with 0.5 ml 0.9% NaCl, and ¹²⁵I-insulin binding assay conducted, as described previously (29). Briefly, 15,000 dpm ¹²⁵I-insulin (approximately 200 pg/ml) were added directly into the 24-well culture plates with or without unlabeled hormones. Final assay vol was 500 µl PBS (1.6% BSA, pH 8.0). At the end of the 2 h binding assay, wells were washed in PBS and cells were solubilized with 1 N NaOH and placed in 12 x 75-mm tubes. Culture wells were washed twice, and these washes were combined with cells and counted in an automated counter.

Exp 6 was conducted to evaluate whether specific binding of ¹²⁵I-leptin existed on granulosa cells. Granulosa cells from small follicles were cultured for 3 days in the presence of 10% FCS, and then cells were washed and ¹²⁵I-leptin binding assays were conducted directly in the culture wells. Leptin was iodinated, using a chloramine-T method, as previously described for insulin-like growth factor-I (30). For Exp 6a, approximately 100,000 dpm ¹²⁵I-leptin was incubated for 0–6 h at 25 C in a total assay vol of 400 µl (.25% BSA in PBS, pH = 7.5), whereas for Exp 6b, 50,000–400,000 dpm ¹²⁵I-leptin were incubated for 6 h at 25 C in a total assay vol of 400 µl. Specific binding was determined as the difference between total binding and nonspecific binding for both experiments; nonspecific binding was determined using 5 µg/well of unlabeled recombinant mouse leptin.

Determination of granulosa cell numbers
Numbers of granulosa cells were determined at the termination of experiments using a Coulter counter (Model Zm; Coulter Electronics, Hialeah, FL), as previously described (25). Briefly, cells were exposed to 0.5 ml trypsin (.25% [wt/vol] in 0.15 M NaC1) for 20 min at 25 C, then scraped from each well, diluted in 0.15 M NaC1, and enumerated.

Progesterone RIA
Concentrations of progesterone in culture medium collected on day 3 of culture were determined with an RIA, as previously described (25). Intra- and interassay coefficients of variation were 11 and 18%, respectively. Sensitivity of the progesterone assay was 25 pg/tube.

Functional aromatase activity
Functional aromatase activity was assessed during the last 24 h of exposure of granulosa cells to 500 ng/ml testosterone. After the last 24 h incubation, concentrations of estradiol in medium were determined by RIA (26, 28), and cell numbers were determined. Estradiol production was expressed as picograms/10⁵ cell·24 h.

Statistical analyses
Experimental data are presented as the least-squares means ± SE of measurements for triplicate culture wells from two or more experiments. Each experiment was performed with different pools of granulosa cells collected from 20–30 ovaries (X ± SE, 24 ± 2 ovaries) from 10–15 cows for each pool. Main effects (e.g. dose) and interactions on dependent variables (i.e. steroid production) were assessed using general linear models procedure of Statistical Analysis System (31). Each well was a replicate, and each experiment contained three replicates per treatment. When steroid production was expressed as nanograms or picograms/10⁵ cells·24 h, cell numbers at the termination of the experiment were used for this calculation. Specific differences in steroid production between treatments were determined using the Fisher’s protected least significant difference procedure (32).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exp 1
In the absence of leptin, 100 ng/ml insulin increased (P < 0.05) small-follicle granulosa cell numbers by 2.6-fold (Table 1) and increased (P < 0.01) progesterone (Fig. 1A) and estradiol production (Fig. 1B) by 1.6- and 6.5-fold, respectively. Doses of 100 and 300 ng/ml leptin inhibited (P < 0.05) insulin-induced increases in progesterone production by 48% and 82%, respectively, whereas 10 and 30 ng/ml leptin were without effect (P > 0.05). Doses of 10, 30, 100, and 300 ng/ml leptin decreased (P < 0.01) the insulin-induced increases in estradiol production by 31%, 46%, 81%, and 89%, respectively (Fig. 1B). In contrast, leptin did not affect (P > 0.10) insulin-stimulated small-follicle granulosa cell numbers (Table 1).

View larger version (32K): [in this window] [in a new window]   Figure 1. Effects of leptin on insulin-stimulated estradiol (Panel A) and progesterone (Panel B) production by granulosa cells from small (1–5 mm) follicles (Exp 1). Granulosa cells from small follicles were cultured for 2 days in the presence of 10% FCS and then treated in serum-free media with 500 ng/ml testosterone, 50 ng/ml FSH, and 0 (open panels) or 100 ng/ml (hatched panels) of insulin for an additional 24 h. Medium was changed every 24 h. During the last 24 h of culture, leptin (0, 10, 30, 100, and 300 ng/ml) also was added to the medium. Values are means from three separate experiments; within each replicate experiment, each treatment was applied in triplicate culture wells. Within a panel, means without a common superscript differ (P < 0.05).

Exp 2
In the absence of leptin, insulin increased (P < 0.01) large-follicle granulosa cell numbers by 1.8-fold (Table 1) and increased (P < 0.01) progesterone (Fig. 2A) and estradiol (Fig. 2B) production by 1.3- and 30-fold, respectively. At 10 and 30 ng/ml, leptin decreased (P < 0.01) the insulin-induced increases in progesterone production by 58% and 50%, respectively (Fig. 2A). Doses of 100 and 300 ng/ml completely blocked insulin-induced granulosa cell progesterone production (Fig. 2A). At 10 and 30 ng/ml, leptin decreased (P < 0.01) insulin-induced estradiol production 23% and 39%, respectively. Doses of 100 and 300 ng/ml reduced insulin-induced estradiol production by 51% and 70%, respectively. Similar to granulosa cells from small follicles, leptin did not affect (P > 0.10) insulin-induced proliferation of granulosa cells from large follicles (Table 1).

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of leptin on insulin-stimulated estradiol (Panel A) and progesterone (Panel B) production by granulosa cells from large (8 mm) follicles (Exp 2). Granulosa cells from large follicles were cultured for 2 days in the presence of 10% FCS and then treated in serum-free media with 500 ng/ml testosterone, 50 ng/ml FSH, and 0 (open panels) or 100 ng/ml (hatched panels) of insulin for an additional 24 h, as in Fig. 1. During the last 24 h of culture, leptin (0, 10, 30, 100, or 300 ng/ml) was also added to the medium. Values are means of four separate experiments; within each replicate experiment, each treatment was applied in triplicate culture wells. Within a panel, means without a common superscript differ (P < 0.05).

Exp 5
At 25 ng/ml, insulin inhibited (P < 0.05) ¹²⁵I-insulin binding by granulosa cells (Fig. 3). Total mass of ¹²⁵I-insulin added was approximately 200 pg/ml. In contrast, 25 ng/ml leptin had no effect (P > 0.10) on granulosa cell ¹²⁵I-insulin binding (Fig. 3), suggesting that the inhibitory effect of leptin on insulin action is not mediated by direct inhibition of insulin binding.

View larger version (24K): [in this window] [in a new window]   Figure 3. Comparison of leptin and insulin competing for insulin binding, by granulosa cells collected from small follicles (Exp 5). Granulosa cells were cultured for 3 days in the presence of 10% FCS, and then cells were washed and insulin binding assays were conducted, as described in Materials and Methods. Values are means of three separate experiments and are expressed as a percentage of total binding; within each replicate experiment, each treatment was applied in triplicate culture wells. *, Mean differs (P < 0.05) from control (0).

View larger version (15K): [in this window] [in a new window]   Figure 4. Time course of specific 125I-leptin binding sites in granulosa cells (Exp 6a). Granulosa cells were cultured for 3 days in the presence of 10% FCS, and then cells were washed and leptin binding assays were conducted as described in Materials and Methods. Values are means of three separate experiments; within each replicate experiment, each time point was derived from duplicate total binding wells and duplicate nonspecific binding wells.

View larger version (15K): [in this window] [in a new window]   Figure 5. Specific binding of 125I-leptin to bovine granulosa cells as a function of 125I-leptin concentration (Exp 6b). Granulosa cells were cultured as described in Fig. 4. Values are means of three separate experiments; within each replicate experiment, each datum point was derived from duplicate total binding wells and nonspecific binding wells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Similar to previous reports (26, 27, 28), we found that insulin was a potent stimulator of granulosa cell numbers and estradiol production. Also similar to previous reports (25, 26, 27), we found that insulin’s effect on progesterone production was less pronounced than its effect on estradiol production. Moreover, FSH, in the absence of insulin, had little or no effect on progesterone production by granulosa cells cultured from small and large bovine follicles; this observation is consistent with previous reports (25, 26, 27). However, results of the present study show, for the first time, that leptin may directly antagonize insulin’s stimulatory effect on steroidogenesis at the level of the ovary. In the absence of insulin, leptin had little or no effect on granulosa cell steroidogenesis. Because concentrations of leptin in blood of lean and obese women range from 2–10 ng/ml and 10–100 ng/ml, respectively (1, 2, 3, 4, 5), the present results indicate that leptin at physiological concentrations may have a direct impact on ovarian granulosa cell function. Furthermore, results of the present study indicate that leptin can inhibit insulin-induced steroidogenesis from both undifferentiated and differentiated granulosa cells. Although the locus of leptin’s action on steroidogenesis will require further study, results of the present study indicate that the inhibitory effect of leptin on insulin action is not mediated by direct inhibition of insulin binding to its receptor. A recent report indicates that leptin can attenuate tyrosine phosphorylation of the insulin receptor substrate-1 in cultured HepG2 cells (33), but whether this is the mechanism of action of leptin in granulosa cells will require further study. Interestingly, leptin had no effect on basal or insulin-induced increases in granulosa cell numbers in the present study. These latter observations indicate that leptin does not influence the mitotic action of insulin, in spite of the fact that leptin reduces insulin-induced granulosa cell steroidogenesis.

Why leptin has positive effects on reproduction in leptin-deficient mice (14), but negative effects on ovarian cell steroidogenesis in vitro, is unclear. This antinomy may be explained by the possible multiple sites of leptin action on the hypothalamic-pituitary-ovarian axis in vivo. In support of this idea, serum LH and FSH concentrations were increased in ob/ob mice injected with leptin (14), and glutamatergic excitatory postsynaptic current in the arcuate nucleus was reduced with leptin treatment (34).

Results of the present study also suggest that ovarian granulosa cells may have high-affinity receptors for leptin. The receptor for leptin in other tissues has been recently identified (35, 36). Although leptin receptors have not been identified previously in granulosa cells, leptin receptors have been identified in other endocrine tissues (37, 38, 39). Recently, leptin receptor messenger RNA has been identified in adult human testis and ovary tissue (40). In fact, the human ovary had one of the highest levels of leptin receptor messenger RNA (40). Collectively, the present and previous studies indicate that the ovary is a likely target organ for leptin.

In conclusion, the results of the present study indicate that leptin, at physiologic concentrations, directly affects insulin-induced steroidogenesis of granulosa cells. We suggest that normally fluctuating concentrations of leptin in blood may play an important role in communicating the metabolic status of the animal to the reproductive system.

	Acknowledgments

 
We thank Beth Keefer and Tricia Hamilton for technical assistance; Wellington Quality Meats (Wellington, KS) for their generous donations of bovine ovaries; N. R. Mason (Lilly Research Laboratories) for the generous donation of estradiol antiserum; and Paula Cinnamon for secretarial assistance.

	Footnotes

 
¹ Approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported under Project H-2088 and Project HR4–032 from the Oklahoma Center for Advancement of Science and Technology.

Received December 12, 1996.

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				S.J. Adamiak, K. Mackie, R.G. Watt, R. Webb, and K.D. Sinclair Impact of Nutrition on Oocyte Quality: Cumulative Effects of Body Composition and Diet Leading to Hyperinsulinemia in Cattle Biol Reprod, November 1, 2005; 73(5): 918 - 926. [Abstract] [Full Text] [PDF]

				M Zerani, C Boiti, C Dall'Aglio, L Pascucci, M Maranesi, G Brecchia, C Mariottini, G Guelfi, D Zampini, and A Gobbetti Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct J. Endocrinol., May 1, 2005; 185(2): 319 - 325. [Abstract] [Full Text] [PDF]

				C Vinoles, M Forsberg, G B Martin, C Cajarville, J Repetto, and A Meikle Short-term nutritional supplementation of ewes in low body condition affects follicle development due to an increase in glucose and metabolic hormones Reproduction, March 1, 2005; 129(3): 299 - 309. [Abstract] [Full Text] [PDF]

				N R Kendall, C G Gutierrez, R J Scaramuzzi, D T Baird, R Webb, and B K Campbell Direct in vivo effects of leptin on ovarian steroidogenesis in sheep Reproduction, December 1, 2004; 128(6): 757 - 765. [Abstract] [Full Text] [PDF]

				M. Zerani, C. Boiti, D. Zampini, G. Brecchia, C. Dall'Aglio, P. Ceccarelli, and A. Gobbetti Ob receptor in rabbit ovary and leptin in vitro regulation of corpora lutea J. Endocrinol., November 1, 2004; 183(2): 279 - 288. [Abstract] [Full Text] [PDF]

				J. E. Swain, R. L. Dunn, D. McConnell, J. Gonzalez-Martinez, and G. D. Smith Direct Effects of Leptin on Mouse Reproductive Function: Regulation of Follicular, Oocyte, and Embryo Development Biol Reprod, November 1, 2004; 71(5): 1446 - 1452. [Abstract] [Full Text] [PDF]

				C. Garofalo, D. Sisci, and E. Surmacz Leptin Interferes with the Effects of the Antiestrogen ICI 182,780 in MCF-7 Breast Cancer Cells Clin. Cancer Res., October 1, 2004; 10(19): 6466 - 6475. [Abstract] [Full Text] [PDF]

				M. L. Hamm, G. K. Bhat, W. E. Thompson, and D. R. Mann Folliculogenesis Is Impaired and Granulosa Cell Apoptosis Is Increased in Leptin-Deficient Mice Biol Reprod, July 1, 2004; 71(1): 66 - 72. [Abstract] [Full Text] [PDF]

				A. Matsunuma, T. Kawane, T. Maeda, S. Hamada, and N. Horiuchi Leptin Corrects Increased Gene Expression of Renal 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase and -24-Hydroxylase in Leptin-Deficient, ob/ob Mice Endocrinology, March 1, 2004; 145(3): 1367 - 1375. [Abstract] [Full Text] [PDF]

				N. K. Ryan, K. H. Van der Hoek, S. A. Robertson, and R. J. Norman Leptin and Leptin Receptor Expression in the Rat Ovary Endocrinology, November 1, 2003; 144(11): 5006 - 5013. [Abstract] [Full Text] [PDF]

				M. Archanco, F. J. Muruzabal, D. Llopiz, M. Garayoa, J. Gomez-Ambrosi, G. Fruhbeck, and M. A. Burrell Leptin Expression in the Rat Ovary Depends on Estrous Cycle J. Histochem. Cytochem., October 1, 2003; 51(10): 1269 - 1277. [Abstract] [Full Text] [PDF]

				C. C. Francisco, L. J. Spicer, and M. E. Payton Predicting Cholesterol, Progesterone, and Days to Ovulation Using Postpartum Metabolic and Endocrine Measures J Dairy Sci, September 1, 2003; 86(9): 2852 - 2863. [Abstract] [Full Text] [PDF]

				N. Kado, J. Kitawaki, H. Koshiba, H. Ishihara, Y. Kitaoka, M. Teramoto, and H. Honjo Relationships between the serum levels of soluble leptin receptor and free and bound leptin in non-pregnant women of reproductive age and women undergoing controlled ovarian hyperstimulation Hum. Reprod., April 1, 2003; 18(4): 715 - 720. [Abstract] [Full Text] [PDF]

				M. Caprio, E. Fabbrini, G. Ricci, S. Basciani, L. Gnessi, M. Arizzi, A. R. Carta, M. U. De Martino, A. M. Isidori, G. V. Frajese, et al. Ontogenesis of Leptin Receptor in Rat Leydig Cells Biol Reprod, April 1, 2003; 68(4): 1199 - 1207. [Abstract] [Full Text] [PDF]

				Z. T. Ruiz-Cortes, Y. Martel-Kennes, N. Y. Gevry, B. R. Downey, M.-F. Palin, and B. D. Murphy Biphasic Effects of Leptin in Porcine Granulosa Cells Biol Reprod, March 1, 2003; 68(3): 789 - 796. [Abstract] [Full Text] [PDF]

				P. Cameo, P. Bischof, and J. C. Calvo Effect of Leptin on Progesterone, Human Chorionic Gonadotropin, and Interleukin-6 Secretion by Human Term Trophoblast Cells in Culture Biol Reprod, February 1, 2003; 68(2): 472 - 477. [Abstract] [Full Text] [PDF]

				R. P. Wettemann, C. A. Lents, N. H. Ciccioli, F. J. White, and I. Rubio Nutritional- and suckling-mediated anovulation in beef cows J Anim Sci, February 1, 2003; 81(14_suppl_2): E48 - 59. [Abstract] [Full Text] [PDF]

				C. C. Francisco, C. S. Chamberlain, D. N. Waldner, R. P. Wettemann, and L. J. Spicer Propionibacteria Fed to Dairy Cows: Effects on Energy Balance, Plasma Metabolites and Hormones, and Reproduction J Dairy Sci, July 1, 2002; 85(7): 1738 - 1751. [Abstract] [Full Text] [PDF]

				N. K. Ryan, C. M. Woodhouse, K. H. Van der Hoek, R. B. Gilchrist, D. T. Armstrong, and R. J. Norman Expression of Leptin and Its Receptor in the Murine Ovary: Possible Role in the Regulation of Oocyte Maturation Biol Reprod, May 1, 2002; 66(5): 1548 - 1554. [Abstract] [Full Text]

K. Kume, K. Satomura, S. Nishisho, E. Kitaoka, K. Yamanouchi, S. Tobiume, and M. Nagayama Potential Role of Leptin in Endochondral Ossificati