Doctoral Dissertations

Date of Award

8-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Animal Science

Major Professor

James D. Godkin

Committee Members

Linda Munson, Arnold M. Saxton, Lilitha C. Mendis-Handagama, F. Neal Schrick

Abstract

Study 1. An experiment was carried out to determine whether peripheral hormone concentrations quantified between 2 and 12 months (mo) were meaningfully correlated with testis size and number of Sertoli and germ cells in the yearling beef bull. Twenty four Angus bulls were treated monthly with GnRH (0.05 μg per Kg of body weight) and bled 1.5 and 3 hours (h) later. On the day before the GnRH challenge, three blood samples were taken at 1.5-h intervals to establish basal hormone concentrations. At 12 mo, all bulls were surgically castrated. Concentrations of FSH, LH, testosterone (T), androstenedione (δ4A) and 17β-estradiol (E2) were quantified by RIA and number of Sertoli cells per testis and quantitative aspects of spermatogenesis were determined. Analysis of regression and Pearson's partial correlation were used to estimate the degree of association between hormone and testis data. Histology analysis revealed that 3 bulls had no spermatids and, therefore, were infertile. Yearling testis weight (TW) was related to GnRH-stimulated T at 3 mo (r = 0.48, p < 0.05) and E2 and 2 and 4 mo (r = 0.40 and 0.45, p < 0.10). Basal and GnRH-stimulated FSH quantified between 2 and 12 mo were correlated with TW (r = - 0.37, p < 0.10 to - 0.71, p < 0.01), number of Sertoli cells (r = nonsignificant, p > 0.10 to r = -0.59, p < 0.05) and number of round spermatids per testis (r = nonsignificant, p > 0.10 to - 0.69, p < 0.01). Also, FSH between 2 and 12 mo correlated with number of round spermatids per A1 spermatogonia (r = nonsignificant, p > 0.10 to r = - 0.61, p < 0.01), number of round spermatids per Sertoli cell (r=nonsignificant, p > 0.10 to - 0.64, p < 0.01) and percent of seminiferous tubules with round spermatids (r = nonsignificant, p > 0.10 to r = - 0.63, p < 0.01) in the 12-mo old testis. Regression analysis showed that FSH and E2 at 2 mo accounted for 45% of the variation in TW. Furthermore, significant variation in TW was accounted by FSFI and testis diameter at 6 mo (R2 = 0.57), FSH and testis diameter at 9 mo (R2 = 0.67) and FSH and body weight at 12 mo (R2 = 0.63). Thus, it appears that prepubertal testis size and FSH could be used as indicators of testicular development of yearling beef bulls. However, the inclusion of data from infertile bulls in the correlation and regression analysis may have generated biased results. Three cases of spermatogenic arrest were described in this study. Bull #250 had seminiferous tubules lined by Sertoli cells only and bull #345 had no germ cells that advanced beyond the A1 spermatogonia stage. Testes of these two bulls weighed 60% less than those of normal bulls (p < 0.01). Concentrations of T and LH in bulls #250 and #345 were reduced at some ages (p < 0.10). Spermatogenic arrest at the level of pachytene spermatocyte was detected in bull #389. Basal FSH and LH were higher in bull #389 than in normal bulls (p < 0.05). Testes of this bull weighed 13% of those of normal bulls and secreted less T throughout the year (p < 0.05). However, as in the cases of bulls #250 and #345, the decrease in T observed in bull#389 did not appear to be sufficient to interrupt germ cell development. Chromosomal abnormalities and/or deficiency in intratesticular factors may be the causes of infertility in these bulls.

Study 2. The objective of this study was to determine whether concentrations of gonadotropins and steroids in 2- and 6-month old beef bulls were significantly correlated with testicular development and number of Sertoli cells at the age of 6 months (mo). At 2 mo, 15 crossbred bulls were treated with GnRH (5 μg) and bled 1.5, 3 and 4.5 hours (h) later. One day before the GnRH challenge, three basal samples were taken at 1.5-h intervals. At 6 mo, bulls received 1 mg of estradiol benzoate (E) and were treated with GnRH (10 pg) 12 h later. After 5 days, bulls received GnRH only (10 pg). In both cases, blood samples were taken 1.5, 3 and 4.5 h after GnRH and two basal samples were taken 1 h before the GnRH treatment. Bulls were castrated at 6 mo. Concentrations of FSH, LH, testosterone (T), androstenedione (δ4A) and 17β-estradiol (E2) were quantified by RIA. Number of Sertoli cells and A1 spermatogonia per testis and quantitative aspects of spermatogenesis were also estimated. Analysis of regression and Pearson's partial correlation were used to estimate the degree of association between hormone and testis data. Results showed that testis weight at 6 mo (TW) was related to FSH at 2 mo (r = - 0,48 to - 0.62, p = 0.08 to 0.02). Also, concentrations of FSH at 2 mo was correlated with number of Sertoli cells (SC; r = - 0.50 to - 0.65, p = 0.07 to 0.01), A1 spermatogonia (Al; r = - 0.45 to - 0.58, p = 0.09 to 0.03) and number of seminiferous tubules with round spermatids (r = - 0.49 to - 0.60, p = 0.08 to 0.02). Regression analysis showed that FSH and body weight at 2 mo accounted for 30% and 72% of the variation in SC and TW in the 6-mo old bull. Concentrations of estradiol at 6 mo were related to SC (r = 0.62, p = 0.02). At 6 mo, GnRH-stimulated FSH and LH were respectively 2.5- and 3-fold higher when GnRH was given in combination with E than when GnRH was given alone. Correlation between FSH and SC was more pronounced after GnRH+E (r = -0.68, p = 0.008) than after GnRH only (r = - 0.63, p = 0.015). Regression modeling showed that FSH and body weight at 6 mo accounted for 48% of the variation in the population of Sertoli cells. Thus, concentrations of FSH at 2 and 6 mo of age appears to be reliable indicators of number of Sertoli cells and testicular development in the 6-mo beef bull.

Study 3. This study was carried out to determine whether testis size, histology and hormone secretion at the developmental state at which concentrations of testosterone reached 1 ng/ml of serum were related to size of the testis and quantitative aspects of spermatogenesis in the yearling bull testis. Three basal blood samples (at 1.5-hour intervals) were taken from 28 Angus calves since the age of 15 weeks (wk). When concentrations of testosterone (T) reached 1 ng/ml, bulls were unilaterally castrated. On a monthly base, bulls received 0.05 pg of GnRH per Kg of body weight and were bled 1.5 and 3 hours later. At 54 wk, the second testis was excised. RJA's were used to quantify FSH, LH, T, androstenedione (δ4A) and 17β-estradiol (E2) in blood samples. Number of Sertoli cells and quantitative aspects of spermatogenesis were estimated in both testes. Analysis of regression and Pearson's partial correlation were used to estimate the degree of association between hormone and testicular development. Results showed that testis weight when T = 1 ng/ml (TWl) was correlated with testis weight (TW2; r = 0.58, p < 0.01), and number of Sertoli cells and round spermatids per testis at 54 wk (r = 0.58, p < 0.01). Also, TWl was related to number of spermatids per Sertoli cell (r = 0.41, p < 0.05), and number of seminiferous tubules with elongate (STE) and mature (STM) spermatids (r = 0.47 and 0.52, respectively; p < 0.05) in the second testis. However, neither STE nor STM were correlated with number of tubule sections with germ cells in the first testis (STGl). Testis diameter averaged between the ages of 30 and 44 wk (AVTD) and TW2 were correlated with AGEl (r = - 0.58 and - 0.42, p < 0.01), TWl (r = 0.65 and 0.58, p < 0.01) and STGl (r = 0.43 and 0.41, p < 0.10). Regression analysis showed that AGEl and body weight when T = 1 ng/ml accounted for 41% of the variation in testis diameter between 30 and 44 wk. GnRH-stimulated FSH at 11 wk was related to STGl, TWl, TW2 and AVTD (r = - 0.42 to - 0.53, p < 0.05). GnRH-stimulated T at 15 wk was related to TWl (r = 0.55, p < 0.01) and AVTD (r = 0.48, p < 0.05). Also, bulls that secreted more E2 after a GnRH treatment at 15 wk had larger AVTD (r = 0.62, p < 0.62, p < 0.01) and TW2 (r = 0.45, p < 0.10). Thus, differences between slow and fast growing bulls are affected by the ability of the testis to secrete testosterone early in life. Also, the existence of correlations between FSH, steroids and characteristics of the testis at AGEl and prepuberty points toward a role played by these hormones at distinct phases of germ cell development.

Study 4. A study was conducted to identify the cell types that express retinol-binding protein (RBP) in the bovine testis and to evaluate the profiles of RBP mRNA levels as related to testicular development. Furthermore, the hypothesis that RBP could serve as a marker of state of development of the seminiferous epithelium was evaluated. At the ages of 10 (n=3), 20 (n=8) and 34 (n=7) weeks, Angus bulls were bled three times and surgically castrated afterwards. Blood samples were assayed for LH and testosterone (T) and the degree of seminiferous tubule development was evaluated in tissue samples stained with hematoxylin and eosin. Immunolocalization of RBP was detected according to the biotin-strepavidinhorseadish peroxidase method (samples from 10-wk old testes could not be analyzed). Nylon membranes containing isolated total RNA were hybridized with a bovine cDNA (bcRBP-700) and a β-actin probe and slot blots were carried out to determine the abundance of RBP and β-actin transcripts. Age-related changes in testis weight, hormone concentrations and relative quantity of RBP transcripts were determine by analysis of variance. Results showed that testis weight and levels of LH and T increased with age (p < 0.05). Also, seminiferous tubules at 10 wk had immature Sertoli cells and gonocytes but, at 20 wk, spermatogonia and few spermatocytes were detected. At 34 wk, Sertoli cells appeared differentiated and spermatids were observed. As never shown before in the bovine species, RBP was detected in Sertoli, Leydig and peritubular cells at the ages of 20 and 34 wk, but no immunoreactivity was present in the germ cells. Furthermore, no difference in staining between Sertoli cells from tubules with or without germ cells was detected. Thus, as determined by immunohistochemistry, RBP does not appear to be a distinct marker of Leydig and Sertoli cell differentiation. Northern hybridization of testicular RNA revealed the presence of a mlUSfA of 1.1 Kb, which was similar to previous RBP transcripts found in bovine conceptuses and extraembryonic membranes. Densitometric scanning of slot blots indicated that the fraction of total RNA coding for RBP was 50 % higher at 10 wk than at 20 and 34 wk. Thus, it is possible that testicular RBP mRNA is down-regulated by testosterone and/or LH, but further studies are needed to verify this hypothesis.

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