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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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INSL3 protein expression in selected macaque tissues. a. Western blot analysis of INSL3 in selected endocrine-reproductive tissues. Eighty μg of total protein isolated from the ovary (Ov), testis (Te), hypothalamus (Hyp) and pituitary (Pit) was loaded in each lane. β-ACTIN (~47 kDa) was used as an internal loading control, and PageRuler™ Plus Prestained Protein Ladder (Fermentas) was used as molecular weight marker (MW). b. Immunohistochemistry (IHC) detection of INSL3 protein in the macaque ovary. Positive signal (brown) of INSL3 was localized in the thecal cells (The) surroundingantral follicles (AF), but not in other cell types within the ovary (A, B). Normal rabbit serum stained ovary section was used as negative control (C). GC, granulosa cells. c. INSL3 levels in monkey sera and follicular fluid during controlled ovarian stimulation (COS) protocols at 0-h and 36-h post hCG treatment. F serum follicular, female serum at follicular phase; F serum luteal, female serum at luteal phase; M, male. The value is presented as mean ± SE. Tissue lysate, sections, blood and follicular fluid were isolated from 3-4 different animals.
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Figure 3: INSL3 protein expression in selected macaque tissues. a. Western blot analysis of INSL3 in selected endocrine-reproductive tissues. Eighty μg of total protein isolated from the ovary (Ov), testis (Te), hypothalamus (Hyp) and pituitary (Pit) was loaded in each lane. β-ACTIN (~47 kDa) was used as an internal loading control, and PageRuler™ Plus Prestained Protein Ladder (Fermentas) was used as molecular weight marker (MW). b. Immunohistochemistry (IHC) detection of INSL3 protein in the macaque ovary. Positive signal (brown) of INSL3 was localized in the thecal cells (The) surroundingantral follicles (AF), but not in other cell types within the ovary (A, B). Normal rabbit serum stained ovary section was used as negative control (C). GC, granulosa cells. c. INSL3 levels in monkey sera and follicular fluid during controlled ovarian stimulation (COS) protocols at 0-h and 36-h post hCG treatment. F serum follicular, female serum at follicular phase; F serum luteal, female serum at luteal phase; M, male. The value is presented as mean ± SE. Tissue lysate, sections, blood and follicular fluid were isolated from 3-4 different animals.

Mentions: Using an antibody raised against human INSL3 precursor, an approximately 14.5 kDa band was detected in the macaque ovary, testis and pituitary (Figure 3a). The size of macaque INSL3 is identical to that of mouse (not shown). IHC detected specific staining of INSL3 antibody only in the theca layers surrounding antral follicles in the ovary (Figure 3b). Since only part of each macaque tissue was used for protein extraction and Western blot, quantitative analysis of the Western blot was not performed due to the uneven distribution of INSL3-expressing follicles in the ovary.


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)

INSL3 protein expression in selected macaque tissues. a. Western blot analysis of INSL3 in selected endocrine-reproductive tissues. Eighty μg of total protein isolated from the ovary (Ov), testis (Te), hypothalamus (Hyp) and pituitary (Pit) was loaded in each lane. β-ACTIN (~47 kDa) was used as an internal loading control, and PageRuler™ Plus Prestained Protein Ladder (Fermentas) was used as molecular weight marker (MW). b. Immunohistochemistry (IHC) detection of INSL3 protein in the macaque ovary. Positive signal (brown) of INSL3 was localized in the thecal cells (The) surroundingantral follicles (AF), but not in other cell types within the ovary (A, B). Normal rabbit serum stained ovary section was used as negative control (C). GC, granulosa cells. c. INSL3 levels in monkey sera and follicular fluid during controlled ovarian stimulation (COS) protocols at 0-h and 36-h post hCG treatment. F serum follicular, female serum at follicular phase; F serum luteal, female serum at luteal phase; M, male. The value is presented as mean ± SE. Tissue lysate, sections, blood and follicular fluid were isolated from 3-4 different animals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: INSL3 protein expression in selected macaque tissues. a. Western blot analysis of INSL3 in selected endocrine-reproductive tissues. Eighty μg of total protein isolated from the ovary (Ov), testis (Te), hypothalamus (Hyp) and pituitary (Pit) was loaded in each lane. β-ACTIN (~47 kDa) was used as an internal loading control, and PageRuler™ Plus Prestained Protein Ladder (Fermentas) was used as molecular weight marker (MW). b. Immunohistochemistry (IHC) detection of INSL3 protein in the macaque ovary. Positive signal (brown) of INSL3 was localized in the thecal cells (The) surroundingantral follicles (AF), but not in other cell types within the ovary (A, B). Normal rabbit serum stained ovary section was used as negative control (C). GC, granulosa cells. c. INSL3 levels in monkey sera and follicular fluid during controlled ovarian stimulation (COS) protocols at 0-h and 36-h post hCG treatment. F serum follicular, female serum at follicular phase; F serum luteal, female serum at luteal phase; M, male. The value is presented as mean ± SE. Tissue lysate, sections, blood and follicular fluid were isolated from 3-4 different animals.
Mentions: Using an antibody raised against human INSL3 precursor, an approximately 14.5 kDa band was detected in the macaque ovary, testis and pituitary (Figure 3a). The size of macaque INSL3 is identical to that of mouse (not shown). IHC detected specific staining of INSL3 antibody only in the theca layers surrounding antral follicles in the ovary (Figure 3b). Since only part of each macaque tissue was used for protein extraction and Western blot, quantitative analysis of the Western blot was not performed due to the uneven distribution of INSL3-expressing follicles in the ovary.

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
Related in: MedlinePlus