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Graduate Program

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Graduate Faculty Program Description

OOCYTE MATURATION AND EMBRYO DEVELOPMENT
IN DOMESTIC ANIMALS

Charlotte E. Farin
Char_Farin@ncsu.edu

OVERALL OBJECTIVE

The overall objective of research conducted in my laboratory is to support the development of in vitro systems for the production of livestock embryos by investigating (1) mechanisms controlling the maturation of mammalian oocytes; and (2) mechanisms affecting embryo and fetal development.

Understanding mechanisms regulating the resumption of meiosis in mammalian oocytes supports the development of methods for manipulating the process of meiotic maturation and improving oocyte developmental competence. Understanding the regulation of fetal development supports the development of methods for identifying normal and abnormal embryos resulting from in vitro production procedures. 

Together, information on oocyte maturation and embryo development can be used to improve the effectiveness of procedures for assisted reproductive technologies in domestic species.

DESCRIPTION OF ONGOING RESEARCH

Bovine, murine and feline oocyte maturation.  Mechanisms regulating maturation of mammalian oocytes are not well understood, therefore we are pursuing several projects designed to investigate the molecular control of bovine, murine and feline oocyte maturation.

We are exploring the requirement for new gene transcription in control of the resumption of meiosis in bovine and feline oocytes cultured in vitro.  This process is also known as germinal vesicle breakdown (GVBD).  Two inhibitors of RNA polymerase II, alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), have been utilized in these studies.  In the presence of DRB and alpha-amanitin, the resumption of meiosis is consistently inhibited in cultured bovine, feline and murine cumulus-oocyte complexes (COC).  This model has been characterized in terms of gonadotropin requirements for maintenance of the DRB-induced block, reversibility of the meiotic inhibition, and assessment of the time-course of sensitivity of GVBD to transcriptional inhibition. 

Our results indicate that the transcriptional pathway leading to meiotic resumption in cultured bovine, murine and feline COC is driven by follicle stimulating hormone, but not luteinizing hormone or human chorionic gonadotropin.  Cumulus cells are required to mediate this transcriptional pathway and key transcriptional events occur within 30 minutes to 1 hour of the initiation of COC culture in cattle and within approximately 20 minutes in mice. 

In an effort to specifically identify transcripts associated with the resumption of meiosis, experiments have been pursued using our DRB model coupled with differential mRNA display (cattle), serial analysis of gene expression (mice) and short interfering RNA (siRNA, cattle), to identify and functionally characterize mRNA species whose expression is associated with the initiation of oocyte maturation in cultured COC. 

We have also collaborated on studies examining the compentency of bovine oocytes recovered from persistent  preovulatory follicles to undergo in vitro maturation, fertilization and development to the blastocyt stage in an effort to understand mechanisms underlying the reduction in fertility in cattle with persistent follicles. 

Other collaborations have included examination of the effect of Vitamin A exposure on oocyte maturation in swine and the effect of female age on oocyte metabolic rates and in vitro embryo development in an endangered felid species, the Pallas’ cat. 

Bovine embryo development.  In vitro production systems are required to produce large numbers of viable embryos for  commercial embryo transfer and for biotechnical manipulations. 

We have examined the developmental competence of embryos produced by current in vitro systems and compared this to in vivo produced embryos.  In vitro produced embryos of morphological quality grade 1 (excellent) are equally capable of establishing pregnancy as in vivo produced embryos of quality grades 1 (excellent) and 2 (good). 

Interestingly, at 7 months of gestation, fetuses resulting from in vitro produced embryos are 17% larger than fetuses from in vivo produced embryos.  Furthermore, as early as 17 days of gestation, elongating embryos resulting from in vitro produced embryos are twice as long as those from in vivo produced embryos. 

We have characterized growth patterns for fetuses derived from in vitro and in vivo production systems, examined the expression of mRNAs for factors known to influence fetal and neonatal growth (IGF-I, IGF-II, their receptors and binding proteins) and have pursued studies of the ultrastructural morphology of bovine embryos produced both in vivo and in vitro. 

We have also completed studies examining various media systems for culture of in vitro produced bovine embryos.  These include Menezo's medium, serum restriction during in vitro culture and defined media.  In addition, we have used the bovine in vitro fertilization and culture system as a diagnostic tool to assess the effect of low-dose progestogen synchronization treatment on oocyte developmental compentency and the effect of exposure to anthelmentics or their metabolites on bovine embryo development.

SELECTED PUBLICATIONS

Farin CE, Alexander JE, Farin PW. 2010. Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro. Theriogenology (accepted). 

Wilson EB, Jordan C, Farin CE. 2010. The successful development of an integrated biotechnology course: challenges faced and lessons learned. NACTA/SERD Conference, Penn State University, June 22-25, 2010 (accepted).

Farin CE, Farmer WT, Farin PW. 2010. Pregnancy recognition and abnormal offspring syndrome. Reprod Fertil Dev 22:75-87. 

Wrench N, Pinto CRF, Klinefelter GR, Dix DJ, Flowers WL, Farin CE. 2010. Effect of season on fresh and cryopreserved stallion semen. Anim Reprod Sci 119:219-227.

Bing J, Pratt S, Gillen L-A, Farin C. 2009.  Effectiveness of a distance education laboratory in ‘Anatomy of Domestic Animals’. J Anim Sci 87, E-Suppl.2:383 (abst 499).

Hicks JE, Farin, CE. 2009. Candidate mRNAs regulating meiotic resumption in bovine cumulus-oocyte complexes.  Reprod Fertil Dev. 21:221 (abst 247).

Farin PW, Dowdall KM, Hicks JE, Farin CE, Whisnant CS. 2009. Subcutaneous administration of follicle stimulating hormone for superovulation of Holstein cows. Reprod Fertil Dev. 21:243-244 (abst 293).

Farmer WT, Farin PW, Bischoff SR, Alexander JE, Piedrahita JA, Farin CE. 2008. Detection of antisense to Igf2r (Air) in cattle. Reprod Fert Dev 20:81 (abstr 2).

Farin CE, Rodriguez KF, Alexander JE, Hockney JE, Herick JL, Kennedy-Stoskopf S. 2007.  The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro. Anim Reprod Sci 98:97-112.

Whang HS, Hunt MA, Wrench N, Hockney JE, Farin CE, Tonelli A. 2007. Nonoxynol-9-alpha-cyclodextrin inclusion compound and its application for the controlled release of nonoxynol-9 spermicide. J Applied Polymer Sci 106:4104-4109.

Farin CE, Sommer JR, Rodriguez KF, Petters RM, Alexander JE. 2006. Functional analysis using short-interfering (si)RNA of TRAM-6, a novel transcript associated with oocyte maturation in cattle. Reprod Fert Dev 18:269-270 (abst 324).

Wrench N, Pinto CRF, Klinefleter G, Farin CE. 2006. Seasonal expression and pattern of SP22 immunolocalization in stallion semen. Anim Reprod Sci 94:32-35.

Rodriguez KF, Blomberg LA, Zuelke KA, Miles JR, Alexander JE, Farin CE. 2006. Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression. Physiol Genomics 27:318-327.

Farin PW, Piedrahita J, Farin CE. 2006. Errors in development of fetuses and placentas from in vitro-produced bovine embryos. Theriogenology 65:178-191.

Miles JR, Farin CE, Rodriguez KF, Alexander JE, Farin PW. 2005. Effects of embryo culture on angiogenesis and morphometry of bovine placentas during early gestation. Biol Reprod 73:663-671.

Rodriguez KF, Farin CE. 2004.  Gene transcription and oocyte maturation. Reprod Fert Dev 16:55-67.

Farin CE, Farin PW, Piedrahita JA. 2004.  Regulation of conceptus and fetal development following transfer of in vitro produced and cloned embryos.  J Anim Sci 82(E Suppl):E53-E62

Farin PW, Miles JR, Farin CE. 2004. Pregnancy loss associated with embryo technologies in cattle.  23rd World Buiatrics Congress, Quebec, Canada, July 11-16, 2004 34(1):62-64.

Miles JR, Farin CE, Rodriguez KF, Alexander JE, Farin PW. 2004.  Angiogenesis and morphometry of bovine placentas in late gestation from embryos produced in vivo or in vitro. Bio Reprod 71:1919-1926.

Dindot SV, Farin PW, Farin CE, Romano J, Walker S, Long C, Piedrahita JA. 2004. Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses. Biol Reprod 71:470-478.

Rodriguez KF, Farin CE. 2004.  Developmental capacity of bovine cumulus ooctye complexes after transcriptional inhibition of germinal vesicle breakdown. Theriogenology 61:1499-1511.

Rodriguez KF, RM Petters, Crosier AE, Farin CE. 2002. Role of gene transcription and PKA subtype activation in murine oocyte maturation. Reproduction 123:799-806.

Crosier AE, Farin CE, Rodriguez KF, Blondin P, Alexander J, Farin PW. 2002. Development and candidate gene expression in skeletal muscle of bovine fetuses from embryos produced in vivo and in vitro. Biol Reprod 67:401-408.

Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. 2001.  Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 64:1375-1385.

Farin PW, Crosier AE, Farin CE. 2001. Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 55:151-170.

Farin CE, Farin PW, Blondin P, Crosier AE. 2000. Fetal development of in vitro-produced embryos: possible associations with uterine function. Biennial Symposium on Animal Reproduction, Proceedings American Society of Animal Science, 1999.  Available online at: http://www.asas.org/symposia/proceedings/0920.pdf

Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, West MD, Damiani P. 2000. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2:79-90.

Whaley SL, Hedgpeth VS, Farin CE, Martus NS, Jayes CL, Britt JH. 2000. Influence of vitamin A injection before mating on oocyte development, follicular hormones, and ovulation in gilts fed high-energy diets. J Anim Sci 78:1598-1607.

Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. 2000. Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro. Biol Reprod 62:1459-1465.

Blondin P, Farin PW, Crosier AE, Alexander JE, Farin CE. 2000. In vitro production of embryos alters levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after transfer.  Biol Reprod 62:384-389.

Borchert KM, Farin CE, Washburn SP. 1999. Effect of estrous synchronization with a low dose of progestogen on oocyte integrity in beef cattle. J Anim Sci 77:2742-2748.

Farin CE, Hasler JF, Martus NS, Stokes JE, 1997. A comparison of Menezo B2 and TCM-199 media for in vitro production of bovine blastocysts. Theriogenology 48:699-709.

Wolf CJ, Farin CE, 1996. Effect of gonadotropins on the ability of 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) to inhibit germinal vesicle breakdown in bovine oocytes. Theriogenology 46:759-768.

Piscopo, SE, Farin, CE and Bai, SA. 1996. Determination of concentration of albendazole sulfoxide in plasma and uterine fluid of heifers. Am J Vet Res 58:62-65.

Farin PW, Farin CE, 1995.  Transfer of bovine embryos produced in vivo or in vitro: survival and fetal development.  Biol Reprod 52:676-682.

Martus NS, Farin CE, 1994.  Duration of effectiveness of DRB in inhibiting germinal vesicle breakdown during bovine oocyte maturation. Theriogenology 42:1295-1302.

Farin CE, Yang L, 1994.  Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB).  Mol Reprod Dev 37:284-292.

Yang L, Farin CE, 1994.  Identification of complementary DNAs encoding bovine cyclin B and Cdk1/Cdc2.  Gene 141:283-286.

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