Annual changes in cell proliferation and apoptosis and expression of connexin 43 in the testes of domestic seasonal breeding ganders

Małgorzata Gumułka*, Anna Hrabia, Israel Rozenboim

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Molecular mechanisms of seasonal changes in testicular morphology, histoarchitecture, and functions in birds have not been fully elucidated. The aim of the study was to examine the alternations in cell proliferation and apoptosis, these processes-related gene expressions, and gap junction protein (GJA1gene, connexin 43 protein; Cx43) expression and localization in gander testes during annual stages. Testes from domestic ganders (n = 28) in the first reproduction season were obtained at five stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under controlled breeding conditions. Testicular weight, morphometry, and histology (H&E staining) were evaluated, and the following parameters were tested: (1) number of proliferating (PCNA-positive) and apoptotic (TUNEL-positive) cells; (2) mRNA and protein abundances of PCNA, caspase-3, and Cx43 by qRT-PCR and Western blot, respectively; (3) activity of caspase-3 by fluorometric method; and (4) localization of Cx43 by immunofluoresence. Testicular weight was found to decrease by 4-fold at the NB stage with massive depletion of germ cells concomitantly with a reduction of seminiferous tubule (ST) diameter and ST lumen compared to the PR and OR stages. The number of proliferating germ cells was higher at PrB and PR than at the PoB stage, whereas the number of apoptotic cells was higher at PoB and NB compared to PrB and OR. Thus, proliferation-to-apoptosis ratios were lower in PoB and NB than other stages. Moreover, mRNA expression of PCNA exhibited down regulation in these stages compared to PrB and PR. Stage-dependent changes in the Cx43 mRNA level and in the localization pattern in the ST germinal epithelium were observed. Lower abundances of GJA1 transcript during NB and OR than at PoB the stage and irregular distribution of Cx43 protein located near the lumen of ST primarily at PrB and NB compared to the remaining stages were noted. These results suggested that during gander testis development, function, regression, and recrudescence, the interaction between processes of cell proliferation, apoptosis, and changes in the localization of Cx43 protein in the germinal epithelium occurred. The balance between these processes may determine the final functional activity or inactive stage of the testes connected with weight and histoarchitecture changes.

Original languageAmerican English
Pages (from-to)27-39
Number of pages13
JournalTheriogenology
Volume186
DOIs
StatePublished - 1 Jul 2022

Bibliographical note

Funding Information:
Molecular mechanisms of seasonal alterations in testicular morphology, histoarchitecture, and functions in free-living and domestic birds have not been fully elucidated. Thus, it seems reasonable to undertake an additional investigation into the role of both cell proliferation and apoptosis processes in the annual variations in cell number dynamics of the ST epithelium. In some seasonal breeding mammalian species, variation in proliferative potential of testes has been indicated by the expression of proliferating cell nuclear antigen (PCNA) [ 8?10], a polymerase delta cofactor, which is essential for DNA damage repair during DNA synthesis [11]. In domestic birds, changes in the expression of PCNA protein depended on testicular activity during different photoperiodic conditions have been shown in the Japanese quail [12]. In turn, apoptosis is a form of cell death, allowing the controlled removal of unwanted, damaged, or aged cells. The apoptotic event results from the activation of multiple signaling pathways, two of which, the extrinsic (or death receptor pathway) and intrinsic (or mitochondrial), are the most important. Both pathways end at the point of the execution phase mediated by intracellular cysteine proteases (caspases), in which caspase-3 is considered to be the most important and functions as effector (executioner) caspase [13]. Apoptosis has been reported to contribute to germ cell degeneration and seasonal testicular regression in mammalian species like the brown hare [8], South American plains vizcacha, Lagostomus maximus [9], and Japanese field mice [10]. In birds, apoptotic cell death is considered a remarkable process connected with testis development in chicken embryos [14] and the changes in the number of ST epithelial germ cells and spermatogenesis supporting Sertoli cells at a transitional stage of testicular remodeling during the seasonal period [ 2?4]. Moreover, apoptosis is the process that modulates alternation in testicular size in the Japanese quail in different experimental conditions created by pharmacological treatment [15] or photoperiod changes [16].The authors are grateful to Dr. Dominika Wolak from the University of Agriculture in Krakow for valuable help in Western blot analysis and to Justyna Kolasa, M.Sc. for proliferating cell counting. This work was financially supported by the Ministry of Sciences and Higher Education for the University of Agriculture in Krakow, Poland (subvention number: SUB-020013-D015 and SUB-020002-D015).

Funding Information:
This work was financially supported by the Ministry of Sciences and Higher Education for the University of Agriculture in Krakow , Poland (subvention number: SUB- 020013-D015 and SUB-020002-D015 ).

Publisher Copyright:
© 2022 Elsevier Inc.

Copyright © 2022 Elsevier Inc. All rights reserved.

Keywords

  • Apoptosis
  • Connexin 43
  • Ganders
  • Proliferation
  • Seasonal breeding
  • Testes

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