Examination of relaxin and its receptors expression in pig gametes and embryos.
BACKGROUND
Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos.


METHODS
Immature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After in vitro-maturation, COCs were in vitro-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls.


RESULTS
Using both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or in situ immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting.


CONCLUSIONS
All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.
Department of Animal & Dairy Sciences, Mississippi State University, 4025 Wise Center, Mississippi State, MS 38762, USA.
Jean M
JM
Feugang
Juan C
JC
Rodriguez-Munoz
Scott T
ST
Willard
Ross A
RA
Bathgate
Peter L
PL
Ryan
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D054885:Cumulus Cells; D005260:Female; D008297:Male; D009865:Oocytes / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis*; D018000:Receptors, Peptide / Q000096:biosynthesis*; D012065:Relaxin / Q000096:biosynthesis*; D013094:Spermatozoa / Q000378:metabolism*; D034421:Sus scrofa
D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C033395:relaxin receptors; D012065:Relaxin
Theriogenology. 2001 Aug 1;56(3):471-83
11516126
J Clin Endocrinol Metab. 1983 Sep;57(3):451-4
6348059
J Clin Endocrinol Metab. 2005 Jan;90(1):516-21
15483101
Int J Androl. 2001 Feb;24(1):24-30
11168647
J Mol Endocrinol. 1998 Oct;21(2):179-87
9801461
Rev Reprod. 2000 May;5(2):122-30
10864857
J Reprod Dev. 2006 Dec;52(6):773-9
16926527
Hum Reprod. 1997 Jun;12(6):1325-37
9222026
J Biol Chem. 2002 Aug 30;277(35):31283-6
12114498
Ann N Y Acad Sci. 2009 Apr;1160:147-51
19416177
Biol Reprod. 1999 Aug;61(2):512-20
10411534
J Reprod Fertil Suppl. 1993;48:127-38
8145201
Endocrinology. 2008 Mar;149(3):1277-94
18079195
J Reprod Fertil. 1983 Jul;68(2):431-5
6864659
Biol Reprod. 1998 Aug;59(2):401-8
9687314
Biol Reprod. 1980 Sep;23(2):394-7
7417681
Reprod Biol Endocrinol. 2007 Jul 10;5:29
17623071
Reprod Biol Endocrinol. 2004 Jun 16;2:36
15200675
Reproduction. 2006 May;131(5):943-50
16672358
Cell Tissue Res. 2007 Nov;330(2):303-12
17851698
Endocr Rev. 2004 Apr;25(2):205-34
15082520
Proc Natl Acad Sci U S A. 2004 May 11;101(19):7323-8
15123806
Ann N Y Acad Sci. 2005 May;1041:61-76
15956688
Adv Exp Med Biol. 2007;612:34-48
18161480
Hum Reprod Update. 2006 Nov-Dec;12(6):757-67
16882702
FASEB J. 2006 Nov;20(13):2352-62
17077312
J Reprod Fertil. 1980 Mar;58(2):369-75
7431269
J Biol Chem. 2006 Nov 17;281(46):34942-54
16963451
Mol Hum Reprod. 2005 Aug;11(8):591-600
16051677
Ann N Y Acad Sci. 2009 Apr;1160:192-3
19416186
Mol Reprod Dev. 2002 Mar;61(3):414-24
11835587
Endocrinology. 2007 Mar;148(3):1181-94
17158203
Ann N Y Acad Sci. 2005 May;1041:17-21
15956682
Endocrinology. 2009 May;150(5):2419-28
19116340
Ann N Y Acad Sci. 2009 Apr;1160:140-6
19416176
Int J Androl. 2003 Apr;26(2):115-20
12641830
Hum Reprod. 1999 Feb;14(2):338-44
10099975
J Clin Endocrinol Metab. 1990 Jun;70(6):1771-3
2347909
Biochemistry. 2008 Jul 1;47(26):6953-68
18533687
Theriogenology. 2002 Jan 1;57(1):256-73
11775974
Reprod Domest Anim. 2008 Jul;43 Suppl 2:401-6
18638153
Biol Reprod. 1990 Apr;42(4):737-45
2346777
Biol Reprod. 1986 Feb;34(1):139-43
2420376
Adv Exp Med Biol. 2007;612:14-25
18161478
J Pharmacol Exp Ther. 2007 Jan;320(1):281-90
17065365
J Reprod Dev. 2006 Oct;52(5):657-62
16873991
Mol Cell Endocrinol. 1992 Nov;89(1-2):R5-8
1301378