Balhorn Rod, Steger Klaus, Bergmann Martin, Schuppe Hans-Christian, Neuhauser Stefanie, Balhorn Monique C
a Briar Patch Biosciences LLC , Livermore , CA , USA.
b Department of Urology, Pediatric Urology and Andrology, Section Molecular Andrology , Justus Liebig University , Giessen , Germany.
Syst Biol Reprod Med. 2018 Dec;64(6):424-447. doi: 10.1080/19396368.2018.1510063. Epub 2018 Aug 31.
The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male's haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline.
鱼精蛋白的表达以及这些富含精氨酸的小蛋白与DNA的结合，完成了精子细胞染色质重组过程以及雄性单倍体基因组的整体失活，这一过程发生在哺乳动物精子发育的最后阶段。在过去40年里已制备了多种抗鱼精蛋白抗体，但只有少数几种被证明可用于检测鱼精蛋白的存在、确定其在染色质中的表达和沉积时间，以及研究它们在成熟精子细胞和精子中的结构与功能。这项工作的目的是开发另一组单克隆抗体（MAb），这些抗体不仅能识别新的P1和P2鱼精蛋白表位，还能作为免疫组织化学（IHC）试剂，用于检测和鉴定睾丸组织及射出精子中的哺乳动物鱼精蛋白。以天然和合成的人鱼精蛋白作为抗原，产生了38个识别人类鱼精蛋白P1或P2的杂交瘤克隆。通过酶联免疫吸附测定法（ELISA）筛选了12个最佳克隆产生的抗体的选择性，发现其中两个仅识别人类鱼精蛋白P1或P2，而其他一些则与人及小鼠蛋白都结合。一种单克隆抗体识别所测试的每种鱼精蛋白。所有抗体，包括一种识别种马P1和另一种识别种马P2的抗体，都与精子细胞或精子染色质中的天然鱼精蛋白结合。虽然大多数仅标记伸长的精子细胞或精子，但发现有几种抗体也与缺乏鱼精蛋白的细胞的细胞质或细胞核结合，这表明这些单克隆抗体必须识别鱼精蛋白中也存在于其他蛋白质中的表位。合成了13个重叠的人类鱼精蛋白P1肽，随后用于鉴定六种最佳抗体识别的表位。缩写：BSA：牛血清白蛋白；ELISA：酶联免疫吸附测定；HCl：盐酸；IHC：免疫组织化学；i.p：腹腔内；LIS：二碘水杨酸锂；MAb：单克隆抗体；PBS：磷酸盐缓冲盐水。
