Characterization of Anti-Müllerian Hormone (AMH) Gene in Buffaloes and Goats

Abstract

The Anti-Müllerian Hormone (AMH) is a member of the transforming growth factor beta (TGF-β) superfamily, playing a significant role in cell proliferation, differentiation and apoptosis. In females, AMH is secreted throughout their reproductive life span from ovaries, whereas in males it is secreted by gonadal cells at a very early stage of testicular development. AMH is a promising marker of ovarian reserve in women and can be used to measure the female reproductive lifespan. In the present study, we cloned and sequenced the GC rich AMH gene from Indian riverine buffalo (Bubalus bubalis) and goat (Capra hircus). Obtained sequences were compared to the AMH sequences of other mammals, and corresponding amino acid sequences revealed that the caprine and bovine AMH sequences are more closely related to each other than to those of other mammals. Furthermore, we analyzed the chromosomal localization of AMH genes in mammalian species to understand potential syntenic relationship. The AMH gene is localized between the sequences for the SF3A and JSRP1 genes and maintains this precise location in relation to other nearby genes. The dN/dS ratio of AMH gene did not indicate any pressure for either positive or negative selection; thus, the physiological function of the AMH gene in the reproduction of these two ruminant species remains very vital. Similar to other mammals, the AMH gene may be an important indicator for regulating female reproductive biology function in bovine, cetacean, caprine, and camelidae.

Keywords: Anti-Müllerian hormone; Indian water buffalo; fertility; goat; phylogenetic tree; reproduction; syntenic analysis.

Figure 1

Agarose gel stained with ethidium bromide represents AMH gene fragment of 1728 bp from cDNA of buffalo (lane 1–4) and goat (lane 5–8) granulosa cell. Lane M is 1 Kbp DNA marker.

Figure 2

Phylogenetic and syntenic analysis of AMH: the left panel depicts the phylogenetic analysis of AMH amino acids of sixteen mammalian species performed in MegaX. The rooted phylogenetic tree is constructed from Neighbor-Joining method with bootstrap value of 1,000. The middle panel is showing the chromosomal location of AMH gene in respective animals. The right panel shows the syntenic analysis of the AMH gene of these animals. The neighboring genes SF3A2 on the right side and JSRP1 gene on the left side of AMH are represented in different colors. The arrow head represents the orientation of the genes. AMH gene in the middle is shown in blue color where each box representing the exon. The amino acid accession numbers for phylogenetic and syntenic analysis are: NP_000470.3 (Homo sapiens); NP_999475.2 (Sus scrofa); XP_014977898.2 (Macaca mulatta); NP_776315.1 (Bos taurus); NP_001304192.1 (Equus caballus); XP_014701194.1 (Equus asinus); XP_019820099.1 (Bos indicus); XP_005888453.1 (Bos mutus); NP_001295528.1 (Ovis aries); XP_020761609.1 (Odocoileus virginianus); XP_023990190.1 (Physeter catodon); QBZ68774.1 (Bubalus bubalis); QBZ68773 (Capra hircus); XP_026941471.1 (Lagenorhynchus obliquidens); XP_006206642.1 (Vicugna pacos) and XP_031293953.1 (Camelus dromedarius).

Figure 3

AMH is a member of TGF-β superfamily. (A) AMH signals through its own dedicated type II receptor (AMHRII) like other members of TGF-βs. They phosphorylate their type I receptor (AMHRI) and R-Smads (Smad1, 5, and 8). The active R-Smads then interact with co-Smad4 which translocate to the nucleus and induce various effective roles such as cell growth and differentiation, proliferation and apoptosis. (B) The multiple alignment of TGF-β domain in AMH gene consisting 99 amino acids is done using GeneDoc software. The amino acid accession numbers are: NP_000470.3 (Human); NP_999475.2 (Pig); XP_014977898.2 (Macaca); NP_776315.1 (Taurine Cattle); NP_001304192.1 (Horse); XP_014701194.1 (Donkey); XP_019820099.1 (Indicus Cattle); XP_005888453.1 (Yak); NP_001295528.1 (Sheep); XP_020761609.1 (Deer); XP_023990190.1 (Sperm whale); QBZ68774.1 (Buffalo); QBZ68773 (Goat); XP_026941471.1 (Dolphin) and XP_031293953.1 (Arabian Camel).

Figure 4

Graphic representation of dN/dS ratio for selective pressure on evolution of protein. The sites under selection are studied through SLAC method of Datamonkey server. The coding region of AMH gene used for analysis has the following accession numbers: NM_000479.5 (Homo sapiens); NM_214310.3 (Sus scrofa); XM_015122412 (Macaca mulatta); XM_016934640 (Pan troglodytes); NM_173890 (Bos taurus); NM_001317263 (Equus caballus); XM_014845708 (Equus asinus); XM_019964540 (Bos indicus); XM_005888391 (Bos mutus); NM_001308599 (Ovis aries); XM_020905950 (Odocoileus virginianus); XM_006206580 (Vicugna pacos); XM_024134422 (Physeter catodon); XM_027085670 (Lagenorhynchus obliquidens); XM_004441423 (Ceratotherium simum simum); XM_031438093 (Camelus dromedarius); MH479929 (Bubalus bubalis) and MH479928 (Capra hircus).

Figure 5

Alignment of buffalo and goat AMH proteins (QBZ68774 and QBZ68773) with reference AMH proteins (XP_006047877, XP_017906255, XP_017906254 and AFH66811). Goat AMH isoform 1 (XP_017906254.1) with additional 28 amino acids are shown.