Korean J Physiol Pharmacol.  2018 Nov;22(6):637-647. 10.4196/kjpp.2018.22.6.637.

The treatment effect of novel hGHRH homodimer to male infertility hamster

• Zhang XD
• Guo XY
• Tang JX
• Yue LN
• Zhang JH
• Liu T
• Dong YX
• Tang SS

• KMID: 2430086
• DOI: http://doi.org/10.4196/kjpp.2018.22.6.637

Abstract

Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2-8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

Keyword

Autocrine; Chines hamster; Growth hormone releasing hormone; Homodimer; Male infertility

MeSH Terms

Animals
Cricetinae*
Cricetulus
Cyclophosphamide
Fertility
Gonadotropins
Growth Hormone-Releasing Hormone
Humans
In Situ Nick-End Labeling
Infertility
Infertility, Male*
Male
Male*
Pregnancy Rate
Reproduction
Testis
Testosterone
Vacuoles
Cyclophosphamide
Gonadotropins
Growth Hormone-Releasing Hormone
Testosterone

Figure

• 

  Fig. 1 Experimental design schema. The single CPA group was administered CPA i.p. once in a week in the former 5 weeks and saline i.m. twice in a week in the latter 5 weeks. The hMG or Grin groups were administered CPA i.p. once in a week in the former 5 weeks and added hMG or Grin i.m. twice in a week from the fourth week to the tenth one. All the animals were sacrificed after the tenth week.

• 

  Fig. 2 Changes of body weights or organ weights of the hamster models. (A) Body weight analysis, *p<0.05 vs. single CPA group, T test; (B) Organ weight analysis, *p<0.05, **p<0.01, a vs. single CPA, b vs. hMG group, T test.

• 

  Fig. 3 H-E staining of the testicular tissues [(Mag.4×10 times, bar 100 µm, eosin (pink cytoplasm) and hematoxylin (blue nucleus)]. “十” refers to the intralumen in seminiferous tubule. CPA: cyclophosphamide group, “→” points at pathological vacuole; hMG: human menopausal gonadotropin group, “→” points at the annular clearance, “>” points at the broken wall of seminiferous tubule; H-Grin: high Grin dose; M-Grin: middle Grin dose. L-Grin: low Grin dose.

• 

  Fig. 4 Effect of CPA-induced DNA damage in testicular cells. (A) TUNEL staining (Mag.20×10 times, bar 20 µm, yellow-green TUNEL positive cells). “十” refers to the intra-lumen, “→” points at spermatogonium, “>” points at spermatocyte, “>>” points at spermoblast, “†” points at sperm nuclei, and “‡” refers to sperm body and tail; (B) Quantitative estimation of TUNEL-positive cells. p<0.05* or 0.01**, 0.001*** vs. the single CPA (a) or hMG (b) group.

• 

  Fig. 5 Fluorescent staining of FITC-hGHRH(1-44)NH2 peptide in the testis tissues. (A) Picture merged (Mag.20×10 times, bar 20 µm, GHRH receptor (yellow green) and DAPI (blue nucleus). Symbols are the same as those in Fig. 4; (B) Quantitative estimation of fluorescent staining positive cells of FITC-hGHRH(1-44)NH2 peptide. p<0.05* or 0.01** vs. the single CPA (a) or hMG (b) group.

• 

  Fig. 6 Immunofluorescent staining of GHRH receptor protein in testicular tissues. (A) Picture merged [Mag.20×10 times, bar 20 µm, FITC (yellow GHRH receptor) and DAPI (blue nucleus)]. Symbols are the same as those in Fig. 4; (B) Quantitative estimation of positive cells of GHRH receptor immunofluorescent staining. p<0.05* or 0.01** vs. the single CPA (a), or hMG (b) group.

• 

  Fig. 7 Immunofluorescent staining of GH protein in testicular tissues. (A) Picture merged [Mag.40×10 times, bar 10 µm, Cy3 (red GH) and DAPI (blue nucleus)]. Symbols are the same as those in Fig. 4; (B) Quantitative estimation of positive cells of GH immunofluorescent staining. *p<0.05 or **p<0.01 vs. the single CPA (a) or hMG (b) group.

• 

  Fig. 8 Western blotting analysis of testicular GHRH receptor or GH protein. (A) GHRH receptor (47 kDa) and β-Actin (42 kDa) protein were marked. *p<0.05 or **p<0.01 vs. the single CPA group. (B) GH (24 kDa) and β-Actin (42 kDa) protein were marked.

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