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Expression of insulin-like 3 (INSL3) and differential splicing of its receptor in the ovary of rhesus macaques.

Hanna CB, Yao S, Patta MC, Jensen JT, Wu X - Reprod. Biol. Endocrinol. (2010)

Bottom Line: Moreover, the INSL3 level in follicular fluid is 3-4 times higher than that in female serum which remains low throughout the menstrual cycle.The presence of INSL3 and its receptor in the ovary implies a potential role of the ligand-receptor pair in female reproduction in nonhuman primates.However, the existence of multiple splice variants of RXFP2 indicates a very complex nature of the hormone-receptor system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, West Campus, Beaverton, OR 97006, USA.

ABSTRACT

Background: Although insulin-like 3 (INSL3) has been identified in the gonad of both sexes in many species, there are only limited reports on the distribution of INSL3 and its receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2), in the primate ovary. Since the hormone-receptor pair is believed to play a role in female reproduction, investigating the transcription of INSL3/RXFP2 genes and the spatiotemporal expression of INSL3 in the nonhuman primate may shed light on the functional aspects of the system in humans.

Methods: Database mining, molecular and immunological methods were applied.

Results: One single INSL3 transcript and three novel splice variant transcripts of RXFP2 were identified in the ovary of rhesus macaques. While the full-length RXFP2 transcript is barely detectable in granulosa cells during the periovulatory period, INSL3 transcript and protein are highly abundant in theca cells surrounding antral follicles. Moreover, the INSL3 level in follicular fluid is 3-4 times higher than that in female serum which remains low throughout the menstrual cycle.

Conclusions: The presence of INSL3 and its receptor in the ovary implies a potential role of the ligand-receptor pair in female reproduction in nonhuman primates. However, the existence of multiple splice variants of RXFP2 indicates a very complex nature of the hormone-receptor system.

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Identification of RXFP2 splice variants in the ovary (Ov) and uterus (Ut). a. Agrose gel (1%) electrophoresis of PCR amplicons from both tissues. One kb Plus DNA Ladder (MM, molecular marker) (Invitrogen) was used as marker, and PPIA was used as an internal control. DNA was stained with ethidium bromide. The experiment was repeated with ovary and uterus cDNA derived from three monkeys; b. Simplified schematic representation of RXFP2 exons, transcripts and their corresponding encoding RXFP2 protein regions. LDLa, Low Density Lipoprotein Receptor Class A domain; LRR_RI, Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily; 7tm_1, 7 transmembrane receptor; ATG, translation start codon; TAG, translation stop codon.
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Figure 1: Identification of RXFP2 splice variants in the ovary (Ov) and uterus (Ut). a. Agrose gel (1%) electrophoresis of PCR amplicons from both tissues. One kb Plus DNA Ladder (MM, molecular marker) (Invitrogen) was used as marker, and PPIA was used as an internal control. DNA was stained with ethidium bromide. The experiment was repeated with ovary and uterus cDNA derived from three monkeys; b. Simplified schematic representation of RXFP2 exons, transcripts and their corresponding encoding RXFP2 protein regions. LDLa, Low Density Lipoprotein Receptor Class A domain; LRR_RI, Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily; 7tm_1, 7 transmembrane receptor; ATG, translation start codon; TAG, translation stop codon.

Mentions: The 5'-end of macaque RXFP2 mRNA was amplified by 5'-RACE with cDNA derived from the macaque uterus (GU326354), and the protein deduced from RXFP2 coding region shares 96% identity with human RXFP2 protein (NP_570718.1). Macaque RXFP2 mRNA consists of 18 exons. To demonstrate whether RXFP2 splice variants exist in female reproductive system of rhesus macaques, we amplified exons 6 to 17 in the ovary and uterus by RT-PCR (Figure 1a). To our surprise, besides the full-length transcript at ~1.1 kb, three distinctive splice variants were also amplified from both tissues: RXFP2-sv1 is 877 bp long with exon 15 missing; RXFP2-sv2 is 808 bp and lacks both exons 11 and 15; and the shortest splice variant, RXFP2-sv3 (738 bp), has deleted exons 8, 11 and 15 (Figure 1b). The GenBank accession numbers for three spliced transcripts are GU326355, GU326356 and GU326357, respectively.


Expression of insulin-like 3 (INSL3) and differential splicing of its receptor in the ovary of rhesus macaques.

Hanna CB, Yao S, Patta MC, Jensen JT, Wu X - Reprod. Biol. Endocrinol. (2010)

Identification of RXFP2 splice variants in the ovary (Ov) and uterus (Ut). a. Agrose gel (1%) electrophoresis of PCR amplicons from both tissues. One kb Plus DNA Ladder (MM, molecular marker) (Invitrogen) was used as marker, and PPIA was used as an internal control. DNA was stained with ethidium bromide. The experiment was repeated with ovary and uterus cDNA derived from three monkeys; b. Simplified schematic representation of RXFP2 exons, transcripts and their corresponding encoding RXFP2 protein regions. LDLa, Low Density Lipoprotein Receptor Class A domain; LRR_RI, Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily; 7tm_1, 7 transmembrane receptor; ATG, translation start codon; TAG, translation stop codon.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008693&req=5

Figure 1: Identification of RXFP2 splice variants in the ovary (Ov) and uterus (Ut). a. Agrose gel (1%) electrophoresis of PCR amplicons from both tissues. One kb Plus DNA Ladder (MM, molecular marker) (Invitrogen) was used as marker, and PPIA was used as an internal control. DNA was stained with ethidium bromide. The experiment was repeated with ovary and uterus cDNA derived from three monkeys; b. Simplified schematic representation of RXFP2 exons, transcripts and their corresponding encoding RXFP2 protein regions. LDLa, Low Density Lipoprotein Receptor Class A domain; LRR_RI, Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily; 7tm_1, 7 transmembrane receptor; ATG, translation start codon; TAG, translation stop codon.
Mentions: The 5'-end of macaque RXFP2 mRNA was amplified by 5'-RACE with cDNA derived from the macaque uterus (GU326354), and the protein deduced from RXFP2 coding region shares 96% identity with human RXFP2 protein (NP_570718.1). Macaque RXFP2 mRNA consists of 18 exons. To demonstrate whether RXFP2 splice variants exist in female reproductive system of rhesus macaques, we amplified exons 6 to 17 in the ovary and uterus by RT-PCR (Figure 1a). To our surprise, besides the full-length transcript at ~1.1 kb, three distinctive splice variants were also amplified from both tissues: RXFP2-sv1 is 877 bp long with exon 15 missing; RXFP2-sv2 is 808 bp and lacks both exons 11 and 15; and the shortest splice variant, RXFP2-sv3 (738 bp), has deleted exons 8, 11 and 15 (Figure 1b). The GenBank accession numbers for three spliced transcripts are GU326355, GU326356 and GU326357, respectively.

Bottom Line: Moreover, the INSL3 level in follicular fluid is 3-4 times higher than that in female serum which remains low throughout the menstrual cycle.The presence of INSL3 and its receptor in the ovary implies a potential role of the ligand-receptor pair in female reproduction in nonhuman primates.However, the existence of multiple splice variants of RXFP2 indicates a very complex nature of the hormone-receptor system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, West Campus, Beaverton, OR 97006, USA.

ABSTRACT

Background: Although insulin-like 3 (INSL3) has been identified in the gonad of both sexes in many species, there are only limited reports on the distribution of INSL3 and its receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2), in the primate ovary. Since the hormone-receptor pair is believed to play a role in female reproduction, investigating the transcription of INSL3/RXFP2 genes and the spatiotemporal expression of INSL3 in the nonhuman primate may shed light on the functional aspects of the system in humans.

Methods: Database mining, molecular and immunological methods were applied.

Results: One single INSL3 transcript and three novel splice variant transcripts of RXFP2 were identified in the ovary of rhesus macaques. While the full-length RXFP2 transcript is barely detectable in granulosa cells during the periovulatory period, INSL3 transcript and protein are highly abundant in theca cells surrounding antral follicles. Moreover, the INSL3 level in follicular fluid is 3-4 times higher than that in female serum which remains low throughout the menstrual cycle.

Conclusions: The presence of INSL3 and its receptor in the ovary implies a potential role of the ligand-receptor pair in female reproduction in nonhuman primates. However, the existence of multiple splice variants of RXFP2 indicates a very complex nature of the hormone-receptor system.

Show MeSH