Correlations among ultrasonographic and microscopic characteristics of prepubescent ram lamb testes

Abstract

The onset of spermatogenesis during prepubertal development is accompanied by dynamic changes in testicular microstructure. Computer-assisted analysis of scrotal ultrasonograms may allow us to track these changes in a noninvasive manner; however, the echotextural characteristics of different histomorphological variables remain unclear. Hence the objective of this study was to compare echotextural and microscopic attributes of the testis over the first wave of spermatogenesis in prepubescent ram lambs. Bi-weekly ultrasound examinations and weekly testicular biopsies were carried out in 22 ram lambs from 9.5–10 weeks of age or the attainment of 15 cm³ in testicular volume, respectively, to the first detection of elongated spermatids (ESt). Testicular echogenicity was highly variable with age; however, after the alignment of data to the first detection of ESt, there was an initial increase followed by a decline, corresponding to the mitotic and postmitotic phases of spermatogenesis in prepubescent ram lambs. Testicular echotextural attributes (mean numerical pixel values and pixel heterogeneity) correlated with seminiferous tubule (ST) diameter, the number of degenerating cells/ST cross-section (XS), and the number of ubiquitin C-terminal hydrolase L-1 (a marker for prespermatogonia and undifferentiated spermatogonia) staining cells/ST XS during the mitotic and postmitotic phases. Additionally, in the postmitotic phase, significant correlations were recorded between the quantitative echotextural characteristics and ST cell density, nuclear:ST area and percentages of STs with different spermatogenic cells as the most mature germ cell type present. These results indicate that ram testes exhibit distinctive echotextural characteristics during the mitotic and postmitotic phases of germ cell differentiation. It is concluded that scrotal ultrasonography in conjunction with computerized image analysis holds potential as a noninvasive alternative to testicular biopsy in monitoring the reproductive status throughout different stages of testicular development.

Keywords

Testis ultrasonography echotexture puberty histomorphology spermatogenesis

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References

Kolasa

Misiakiewicz

Marchlewicz

Wiszniewska

. The generation of spermatogonial stem cells and spermatogonia in mammals. Reprod Biol 2012; 12: 5–23.

Sharpe

McKinnell

Kivlin

Fisher

. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction 2003; 125: 769–84.

Orth

Gunsalus

Lamperti

. Evidence from Sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of Sertoli cells produced during perinatal development. Endocrinology 1988; 122: 787–94.

Aitken

Findlay

Hutt

Kerr

. Apoptosis in the germ line. Reproduction 2011; 141: 139–50.

Evans

ACO

Pierson

Garcia

McDougall

Hrudka

Rawlings

. Changes in circulating hormone concentrations, testes histology and testes ultrasonography during sexual maturation in beef bulls. Theriogenology 1996; 46: 345–57.

Chandolia

Bartlewski

Omeke

Beard

Rawlings

Pierson

. Ultrasonography of the developing reproductive tract in ram lambs: Effects of a GnRH agonist. Theriogenology 1997; 48: 99–117.

Chandolia

Honaramooz

Omeke

Pierson

Beard

Rawlings

. Assessment of development of the testes and accessory glands by ultrasonography in bull calves and associated endocrine changes. Theriogenology 1997; 48: 119–32.

Arteaga

Barth

Brito

LFC

. Relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls. Theriogenology 2005; 64: 408–15.

Giffin

Franks

Rodriguez

Hahnel

Bartlewski

. A study of morphological and haemodynamic determinants of testicular echotexture characteristics in the ram. Exp Biol Med (Maywood) 2009; 234: 794–801.

10.

Kauffold

Kessler

Richter

Beynon

Wehrend

. B-mode ultrasound and grey-scale analysis of the epididymis in boars, and the relationship to semen parameters. Reprod Dom Anim 2011; 46: 108–13.

11.

Brito

LFC

Barth

Wilde

Kastelic

. Testicular ultrasonogram pixel intensity during sexual development and its relationship with semen quality, sperm production, and quantitative testicular histology in beef bulls. Theriogenology 2012; 78: 69–76.

12.

Kastelic

Brito

LFC

. Ultrasonography for monitoring reproductive function in the bull. Reprod Dom Anim 2012; 47: 45–51.

13.

Aravindakshan

Honaramooz

Bartlewski

Beard

Pierson

Rawlings

. Gonadotrophin secretion in prepubertal bull calves born in spring and autumn. J Reprod Fertil 2000; 120: 159–67.

14.

Gábor

Sasser

Kastelic

Mézes

Falkay

Bozó

Csík

Bárány

Hidas

Szász

Jr Boros

. Computer analysis of video and ultrasonographic images for evaluation of bull testes. Theriogenology 1998; 50: 223–28.

15.

Wrobel

. The postnatal development of the bovine Leydig cell population. Reprod Dom Anim 1990; 25: 51–60.

16.

Pierson

Adams

. Computer-assisted image analysis, diagnostic ultrasonography and ovulation induction: Strange bedfellows. Theriogenology 1995; 43: 105–12.

17.

Rodriguez-Sosa

Dobson

Hahnel

. Isolation and transplantation of spermatogonia in sheep. Theriogenology 2006; 66: 2091–103.

18.

Gobé

Harmon

. Apoptosis: Morphological criteria and other assays. eLS 2008; 15 Dec: 1–8.

19.

Lunstra

Echternkamp

. Repetitive testicular biopsy in the ram during pubertal development. Theriogenology 1988; 29: 803–10.

20.

Heath

Pugh

Sartin

Navarre

Purohit

. Evaluation of the safety and efficacy of testicular biopsies in llamas. Theriogenology 2002; 58: 1125–30.

21.

Herrera-Alarcón

Villagómez-Amezcua

González-Padilla

Jiménez-Severiano

. Stereological study of postnatal testicular development in Blackbelly sheep. Theriogenology 2007; 68: 582–91.

22.

Salhab

Zarkawi

Wardeh

Al-Masri

Kassem

. Development of testicular dimensions and size, and their relationship to age, body weight and parental size in growing Awassi ram lambs. Small Rumin Res 2001; 40: 187–91.

23.

Steger

Wrobel

. Postnatal development of ovine seminiferous tubules: an electron microscopical and morphometric study. Ann Anat 1996; 178: 201–13.

24.

Ahmadi

Mirshahi

Giffin

Oliveira

MEF

Hahnel

Gao

Bartlewski

. Quantitative relationships between testicular tissue composition and ultrasonographic image attributes in the ram. Vet J 2013; 198: 282–85.

25.

Huang

Guo

Shen

Wang

Chen

Xie

Wang

Zhao

Huo

Lin

Wang

Zhou

Sha

. Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis. J Proteome Res 2008; 7: 3435–46.

26.

Wrobel

Reichold

Schimmel

. Quantitative morphology of the ovine seminiferous epithelium. Ann Anat 1995; 177: 19–32.

27.

Jahnukainen

Chrysis

Hou

Parvinen

Eksborg

Söder

. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol Reprod 2004; 70: 290–6.

28.

Monet-Kuntz

Horcheau-de Reviers

M-T

Terqui

. Variations in testicular androgen receptors and histology of the lamb testis from birth to puberty. J Reprod Fert 1984; 70: 203–10.

29.

Czarnota

Kolios

. Ultrasound detection of cell death. Imaging Med 2010; 2: 17–28.

30.

Tegelenbosch

de Rooij

. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993; 290: 193–200.

31.

Oatley

Brinster

. Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 2008; 24: 263–86.

32.

Luo

Megee

Dobrinski

. Asymmetric distribution of UCH-L1 in spermatogonia is associated with maintenance and differentiation of spermatogonial stem cells. J Cell Physiol 2009; 220: 460–8.

33.

Oatley

Brinster

. The germline stem cell niche unit in mammalian testes. Physiol Rev 2012; 92: 577–95.

34.

Kwon

Mochida

Wang

Y-L

Sekiguchi

Sankai

Aoki

Ogura

Yoshikawa

Wada

. Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis. Biol Reprod 2005; 73: 29–35.

35.

Valasi

Chadio

Fthenakis

Amiridis

. Management of pre-pubertal small ruminants: Physiological basis and clinical approach. Anim Reprod Sci 2012; 130: 126–34.

36.

Skinner

Booth

Rowson

LEA

Karg

. The post-natal development of the reproductive tract of the Suffolk ram, and changes in the gonadotrophin content of the pituitary. J Reprod Fertil 1968; 16: 463–77.

37.

Vlad

Czarnota

Giles

Sherar

Hunt

Kolios

. High-frequency ultrasound for monitoring changes in liver tissue during preservation. Phys Med Biol 2005; 50: 197–213.

38.

Meistrich

Trostle

. Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients. Exp Cell Res 1975; 92: 231–44.