The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT-PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation.

Galactogogues are substances used to induce, maintain, and increase milk production, both in human clinical conditions (like noninfectious agalactias and hypogalactias) and in massification of production in the animal dairy industry. This paper aims to report the state of the art on the possible mechanisms of action, effectiveness, and side effects of galactogogues, including potential uses in veterinary and human medicine. The knowledge gaps in veterinary clinical practice use of galactogogues, especially in the standardization of the lactogenic dose in some pure drugs and herbal preparations, are reviewed.

SummarySirtuin 2 (SIRT2) is a member of a family of NAD +‐dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle‐aged, 13‐month‐old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2 −/− mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis.

Cells of the ovarian follicle undergo extensive proliferation and differentiation from the time that the follicle escapes from the primordial state to its acquisition of ovulatory capacity. We examined the dynamic modification of the phosphorylation state of the histone H3 N-terminal tail in granulosa cells during follicular development. In rodent follicles, the granulosa cell H3 phosphorylation on Ser10 peaks during proestrus. This epigenetic mark is induced by both FSH and 17beta-estradiol (E2), acting independently. E2-induced H3 phosphorylation fails to occur in mice with inactivated alpha-isoform of the nuclear estrogen receptor. E2 induction of histone phosphorylation is attenuated by cell cycle inhibition. Further, E2 induces the activity of the mitotic kinase, Aurora B, in a mammary tumor cell model where mitosis is estrogen receptor-alpha dependent. These results provide evidence for mitotic regulation in follicle development by estrogen and demonstrate a previously undiscovered mechanism for induction of cell proliferation in ovarian and mammary gland cells.

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50-90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation (r = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro.

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT‐PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation. Mol. Reprod. Dev. 56:465–474, 2000. © 2000 Wiley‐Liss, Inc.

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, −F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.