Brief Summary of Minutes of Annual Meeting

Keith Inskeep was the Chair of the meeting since Dr. Milvae could not attend the meeting for medical reasons. Ron Butler nominated Keith Inskeep to act as chair. The meeting was called to order at 9:15 am. Prior to the presentation of reports, Patty Cromack was introduced. She provided background for the MA Experiment Station. The situation of budgets for traveling was discussed. It is clear that there is less funding available to investigators via Hatch and regional projects.

Station reports began with presentations for Objective 1.

NE (Wood): Oocytes were collected from PCOS women with high insulin to test the hypothesis that abnormal oocytes are produced in these patients and this might underlie high abortion in the first trimester. Most of the genes considered to be abnormally expressed were either 1.8- to 10-fold greater than normal. Many of the genes that appear to be abnormal were associated with spindle organization, centrosome, cell-cycle checkpoint and homologous recombination. To study the identified genes, Dr. Wood is trying to develop culture of follicles in vitro from ovaries of 10d-old mice. Gene expression was affected by insulin concentration in the cultures, particularly in the expression of StAR, aromatase, Nek2 and TACC1. Gene expression was also dependent on stage of the estrous cycle. Protein expression also changes during the cell cycle. In another experiment, cows were fed MGA to produce persistent follicles. The cumulus cells were isolated from persistent follicles (16 mm). These will be used to compare gene expression in persistent vs.normal oocytes.

NY (Fortune): The overall question being pursued in this lab is, how does the follicle form? Insulin seems to be responsible for the activation of the follicle, which is the transition from primordial to primary follicle. Culture of follicles with Kit ligand did not produce the same effect as culture with insulin. Insulin caused hypertrophy in the follicles, which was not observed with Kit ligand. Kit ligand also did not support the transition from primary to secondary follicle. The ability to become activated (follicles) appears to be associated with the property to be arrested at the diplotene stage. In large animals, newborns have primary, primordial and antral follicles at birth. Estradiol inhibits activation of the follicles, possibly by delaying the progression to the diplotene stage.

NY (Butler): In this lab, there has been a long-time interest in the relationship of follicle diameter to fertility success in superovulated animals. Generally, moderately larger follicles produce more estradiol and pregnancy is slightly higher. In high producing cows, high metabolic activity and luteal function is observed, but progesterone may be lower because it is rapidly metabolized. This results in less biological effect of progesterone in high producing cows. This lead to the hypothesis that progesterone would be higher and activity of metabolic enzymes lower in pregnant compared to nonpregnant cows. Following timed AI and OVsynch, pregnancy rate did not differ between cows that ovulated a single follicle vs. cows with double ovulations (producing two CL). Cows had greater plasma progesterone than heifers. Nonpregnant and pregnant cows had similar plasma concentrations of progesterone. There was no relationship of pregnancy status with CYP450 RNA or metabolic activity of CYP450 enzyme, which degrades progesterone. Considerable variation was observed among samples in the activity and mRNA of these enzymes, which may make the association with function very difficult.

WI (Wiltbank): Follicles developed after Ovsynch are smaller and produce less estrogen than those developed during a normal cycle. Therefore, the goal of this experiment was to supplement with estradiol. Administration of 1 mg of estradiol closely corresponded to the in vivo concentrations observed in a normal estrous cycle. Manifestation of estrus was improved, but pregnancy rates did not change. However, this seemed to depend on the size of the follicles; estrogen only improves fertility when the follicles are intermediate in size. With ovsynch, almost 50% of the animals did not ovulate or had higher than normal progesterone.

Data in the porcine CL, which does not regress in response to PGF2alpha, suggests that there is a transcription factor that cannot respond to the injection of PGF2alpha. Jun may not be turned on in the pig and this delays the response. This is different than data published using the cow

WV (Yao/Inskeep): Investigations are underway to determine the role of miRNAs in the compromised fertility associated with persistent follicles. miRNA 181a may be associated with the silencing of nucleoplasmin. Nucleoplasmin 2 is important for decondensation of the sperm head, but it needs to be downregulated afterwards. The expression of 181miRNAs matches the degradation pattern of NPM2.

NH (Tsang): Mixed endothelial cells were isolated from a midcycle bovine corpus luteum. MMP-2 activity was identified in this population of cells. TIMP2 is present in the CL, which could interact with the MMPs, forming dimers. In western blots, the molecular weight of TIMP2 was different than expected, which could be due to dimerization with itself or with MMPs.

NH (Townson): The question being addressed is if functional differences exist in endothelial cell subpopulations during different stages of the cycle. No differences in lectin binding to the CL were observed when cells isolated from CL of the cycle vs pregnancy were compared. In addition, expression of several additional proteins was examined, but did not differ between groups.

WV (Flores): Differential gene expression between early and late CL was analyzed using microarrays. Many genes were differentially expressed. Some of the identified geneshave been confirmed with PCR. The same genes also appear to change following administration of PGF2alpha.

Endothelin receptor A may be important to induce luteolysis. Studies using the inhibitor, BQ-610, have produced confounding results, sometimes inhibiting luteolysis while other times not. Other factors may be involved and need to be elucidated.

Objective 2 and related experiments:

WI (Wiltbank): The problem is how to detect animals that are not cycling? Ultrasonography and progesterone analyses are useful to determine if there is a corpus luteum on the ovary. The incidence of anovulation in lactating dairy cows is approximately 25%. Many cows in Ovsynch do not ovulate in the appropriate time, which greatly decreases the rates of conception. Double Ovsynch seems to be more reliable and appears to synchronize and improve conception rates (approximately 50%), which is 15% greater than regular Ovsynch.

MA (Fissore): PLCzeta has been localized to the equatorial region in bull sperm, and its functional characterization continues. Injection activates oocytes to develop to the blastocyst stage.

WV (Inskeep for Bob Dailey): Following proteomics of bull sperm samples, spermadhesin was found to be present in most bulls. However, the isoform 13 only appeared in bulls of low fertility.

Business:
Dr. Joy Pate became new Chair. Jennifer Woods become Director. Rafael Fissore became new secretary. Next meeting will be in Penn State, and Dr. Pate will host the meeting. The time of the meeting remains underdetermined, but it might remain in the first weeks in June. The group appreciates the efforts of Dr. Milvae as past Chair.

At 5:20 pm the meeting was adjourned.

C. O. Lemley, S. T. Butler, W. R. Butler, and M. E. Wilson. Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows. J.Dairy Sci 91 (2):641-645, 2008.

Ferguson DE, Tsang PCW, Schreiber DT, Keator CS, Milvae RA, McCracken JA. 2007. In vivo changes in protein expression of the gelatinases, matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the ovine corpus luteum (CL) after multiple pulses of prostaglandin F2 alpha (PGF2±). The annual meeting of the Society for the Study of Reproduction in San Antonio, Texas; Biol Reprod (special issue): abstract #103, pp.99-100.

Cherry JA, Hou Xiaoying, Rueda BR, Davis JS, Townson DH 2008 Microvascular endothelial cells of the bovine corpus luteum: A comparative examination of the estrous cycle and pregnancy. Journal of Reproduction and Development 54(3): 183-191.

Yoon SY, Fissore RA. Release of phospholipase C zetaand [Ca2+]i oscillation-inducing activity during mammalian fertilization. Reproduction. 2007. 134:695-704.

Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA, Cibelli JB. Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta. BMC Dev Biol. 2008. 8:16.

Wurst A. K., A. B. Dixon, and E. K. Inskeep. 2007. Lack of effect of transrectal ultrasonography with restraint on lambing rate and prolificacy in ewes, Theriogenology 68:1012-1016.

Goravanahally, M. P., A. Sen, E. K. Inskeep, and J.A. Flores. 2007. PKCe and an increase in intracellular calcium concentration are necessary for PGF2± to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells. Reprod. Biol. Endocrinol. 5:37-43.

Lingenfelter, B.M., Dailey, R.A., Inskeep, E.K., Vernon, M.W., Poole, D.H., Rhinehart, J.D. and Yao, J. 2007. Microarray analysis of gene expression in granulosal cells from persistent follicles in cattle. Anim. Reprod. Sci. 104:405-413.

Doerr, M. D, M. P. Goravanahally, J. D. Rhinehart, E. K. Inskeep and J. A. Flores. 2008. Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2&-induced luteolysis of the sheep corpus luteum. Biol. Reprod. 78:688-696.

Wurst, Aimee Kathryn 2007. Effects of Ovulation of Aged Follicles, Pregnancy Diagnosis By Ultrasonography, and Treatments During Lactation On Reproduction in Ewes, Dissertation, West Virginia University, [On-line Abstract].
Available: https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5347

Lingenfelter, Brandon Michael (2008). The Effect of Follicular Aging on Gene Expression in oocytes and Granulosal Cells, Dissertation, West Virginia University, [On-line Abstract].
Available: https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5783

Davis, TL and Pate, JL. 2007. Proliferation of major histocompatibility nonrestricted gamma delta T cells is stimulated by bovine luteal cells. Biol. Reprod. 77:914-922

Hou, X., Arvisais, E.W., Jiang, C., Chen, D, Roy, S.K., Pate, J.L., Hansen, T.R., Rueda, B.R. and Davis, J.S. 2008. Prostaglandin F2± (PGF2±) stimulates the expression and secretion of TGFB1via induction of the early growth response 1 gene (EGR1) in the bovine corpus luteum. Mol. Endocrinol. 22:403-414