Morphometric Evaluation of Spermatogenic Cells and Seminiferous Tubules and Exploration of Luteinizing Hormone Beta Polypeptide in Testis of Datong Yak

Abstract

Histological examination of testes is essential for understanding infertility, sex development, and growth. Therefore, to understand the histomorphology of testes at different developmental stages, we performed hematoxylin and eosin staining of Yak testis. Our results revealed that the diameters of spermatogenic cells and their nuclei were significantly larger (p < 0.05) in the testis at six years compared to at six and 18 months. No significant difference was noted between 30 months and six years. The study was designed to compare the expression profile of LHB in Datong yak. The expression pattern of LHB was explored using quantitative PCR, semi-quantitative PCR, molecular bioinformatic, and Western blot analysis. Our observations indicated that expression of LHB was significantly higher (p < 0.05) in the testis of Datong yak. Western blotting indicated that the molecular mass of LHB protein was 16 kDa in yak. The protein encoded by yak LHB included conserved cysteine-knot domain regions. The high expression of LHB in testis indicated that LHB may be vital for the development of male gonads and the fertility of Datong yak.

Keywords: LHB; gene expression; histomorphology; testis; yak.

Figure 1

Morphological evaluations of testicular tissues at different ages: (A) 6, (B) 18, and (C) 30 months, and (D) 6 years. Different structures were found: myoid cell (My; red arrow), capillary (C), Leydig cell (L), spermatogonium (S; black arrowhead), Sertoli cell (SC; green arrow), primary spermatocyte (PS; Blue arrow), elongated spermatid (ES; Yellow arrow), Seminiferous tubule (ST). Scale bars = 20 µm (10×) and 50 µm (20×).

Figure 2

Analysis of the LHB mRNA using semi-quantitative PCR: (A) expression profiling from various yak tissues; (B) expression profiling at different yak growth stages.

Figure 3

The relative expression of the LHB mRNA level was evaluated by quantitative real-time PCR from different tissues of Datong yak. Different letters indicate a significant difference (p < 0.05).

Figure 4

Assessment of the protein sequence encoded by LHB from the yak. The protein encoded by yak LHB contains a cysteine-knot domain. (A) The sequence of yak LHB and the predicted protein; (B) assessed secondary structures encoded by the LHB protein has a long vertical bar, short vertical bar, medium vertical bar, coil α-helix, extended strand, and sub-medium vertical bar turn; (C) estimated three-dimensional structures of LHB proteins; and (D) prediction of the conserved domain for LHB.

Figure 5

Multiple alignment of the full length sequence of LHB in different animals. Different amino acids are characterized by shaded boxes.

Figure 6

Detection of the expression pattern of LHB protein in Datong yak testis using Western blotting: (A) Western blot of LHB protein and (B) relative LHB protein expression levels were quantified by densitometric analysis. Protein levels were quantified by densitometric analysis. Different letters indicate significant difference (p < 0.05).