Uncovering the Physiological Mechanisms Underlying the Roe Deer (Capreolus capreolus) Testicular Cycle: Analyses of Gelatinases and VEGF Patterns and Correlation with Testes Weight and Testosterone

Abstract

The roe deer (Capreolus capreolus) represents a spontaneous model of testicular inactivation: During winter, bucks show a suspension of spermatogenesis that starts again in spring and peaks during the breeding season (July–August). The underlying mechanisms to the regulation of the cyclic testicular changes are still not fully clear but seem to be imputable to the spermatogenic cell line since other testicular cell populations remain stable without apoptotic phenomena. The aim of the study was to investigate apoptosis, gelatinases (MMP2 and 9), their inhibiting factors (TIMP 1-2), and two isoforms of vascular endothelial growth factor (VEGF121 and 165) with its receptors (VEGFR1-2) in testes collected during pre- and post-rut periods, and to correlate them with testicular weight (TW) and testosterone (TEST). Testes from 18 adult sexually mature bucks were collected in Bologna Apennines (Italy). Samples were weighed and parenchyma collected. Radioimmunoassay, real-time PCR, and zymography were performed. The results showed a post-rut decrease in TW and TEST and an increase in proMMP2, also highlighting a correlation between the gelatinases and the testicular functionality. The VEGF pattern did not show modifications nor correlation with TW and TEST. Overall, gelatinases and their inhibitors, described herein for the first time in roe deer testes, seem to play an important role in the testicular cycle.

Morphological abnormalities in the testes and epididymides of roe deer that affect spermatogenesis

“…-12 at rest; -12 during the rutting period. The selection dates in the rut period (July -August) were chosen based on the authors' research [7][8][9]. The selected material including testes and their epididymides (n=24) was placed in a 10% aqueous solution of neutral formalin for 14 days.…”

Testicular Melatonin and Its Pathway in Roe Deer Bucks (Capreolus capreolus) during Pre- and Post-Rut Periods: Correlation with Testicular Involution

“…Upon death, the scrota with both testes were immediately collected by the personnel of the local biometrical center and refrigerated at 5 ± 1 • C. Within two hours, all samples were moved to the physiology laboratories of Department of Veterinary Medical Sciences of the University of Bologna (Ozzano dell'Emilia, Italy). Ages were assessed upon an analysis of teeth eruption and wear patterns as previously described [35].…”

“…Testicular parenchyma was collected and stored as previously described [35]. Briefly, upon testis isolation, tissue was collected from the middle section, minced, and split into 3 aliquots: two were snap-frozen in liquid nitrogen and stored at −80 • C for hormonal quantification and Western blot analysis; the other one was incubated for one day with an RNA stabilization solution (RNAlater™, Thermo Fisher Scientific, Watham, MA, USA) and stored at −80 • C after solution removal, for gene expression analyses.…”

“…Starting from the end of the rutting period, testes show a morpho-functional involution characterized by a reduction in volume (up to 20%), a reduction in sexual hormones' levels, and a decline of the germ cell line, although without apoptotic events [27][28][29]. Despite several aspects and biomarkers having been investigated, including hormones (FSH, LH, prolactin, estrogens) [26,[30][31][32], androgen receptor distributions [33], growth factors (TGF, IGF, FGF, VEGF) [34], gelatinases (proMMP2) [35], and antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) [36], the processes underlying the annual testicular cycle are still not fully clear. Koziol and colleagues [37] recently reported melatonin concentrations in peripheral blood and the presence of melatonin receptors (MT1 and MT2) in the testis and epididymis of roe deer sampled during different reproductive periods.…”

“…Expression of melatonin receptors MT1 and MT2 was quantified both at protein and messenger RNA levels. Finally, in order to assess changes in the reproductive hormonal profile, the testicular levels of dehydroepiandrosterone (DHEA), an androgen adrenal testosterone prohormone capable of non-sexually related functions [38,39], were quantified and used for a correlation analysis implemented with previously reported data of the same animals [35], such as testis weight and testosterone (T).…”