The combined effects of transient neonatal hypothyroidism and neonatal hemicastration were investigated to see whether they were additive. Hypothyroidism was induced in litters of ten male rats for 25 days from the day of birth by administration of 0.1% (w/v) 6-propyl-2-thiouracil in the mother's drinking water; hemicastration was performed on the day of birth. Controls included both normal and sham-operated animals. Numbers of Sertoli cells and round spermatids were quantified at age 135 days using stereological methods. Sham-operation had no effect on testis mass, or numbers of Sertoli or germ cells. Transient neonatal hypothyroidism resulted in an increase in testicular mass of 27% (P <0.05), whereas neonatal hemicastration resulted in a 33% (P <0.05) increase over control; the combination of the two procedures resulted in a 62% (P < 0.05) increase. There were corresponding significant increases in the number of Sertoli cells: 82% with hypothyroidism, 18% with hemicastration and 123% with the combination of the two procedures. Numbers of round spermatids showed similar increases: 59% with hypothyroidism, 45% with hemicastration and 95% with the combination of the two procedures. It is concluded that the effects of the combination of transient neonatal hypothyroidism and hemicastration are additive with respect to testicular mass, and numbers of Sertoli and germ cells.
D. R. Simorangkir, D. M. de Kretser, and N. G. Wreford
Y. Zhengwei, N. G. Wreford, S. Schlatt, G. F. Weinbauer, E. Nieschlag, and R. I. McLachlan
This study examined the effect of GnRH-antagonist (GnRH-A)-induced gonadotrophin withdrawal on numbers of germ cells in adult cynomolgus monkeys and aimed to identify the site of the earliest spermatogenic lesion(s) produced. Animals received either GnRH-A (Cetrorelix; 450 μg kg−1 day−1 s.c.; n = 5) or vehicle (control, n = 4) for 25 days. One testis was removed on day 16 and the other testis on day 25. The optical disector stereological method was used to estimate germ and Sertoli cell numbers per testis. After GnRH-A treatment for 16 days, the number of type A spermatogonia was unchanged; however, type B spermatogonia (15% of control), preleptotene + leptotene + zygotene (15% control) and pachytene (55% control) spermatocytes were all reduced (P <0.05). By day 25, these cells were further reduced together with step 1–6 spermatids (38% control; P < 0.05). More mature germ cells were unaffected. The proportion of type A pale spermatogonia at stages VII–XII was reduced (P <0.05) in GnRH-A-treated groups (52% on day 16, 43% on day 25) compared with control (67%). After 25 days of GnRH-A treatment, the number of Sertoli cells was unaltered but nuclear volume was reduced (66% control, P < 0.05). Tubule length was unchanged but volume (50% control), diameter (62% control) and epithelial thickness (59% control) were reduced (P < 0.05). GnRH-A treatment suppressed serum testosterone concentrations into the castrate range, and testicular testosterone concentrations to 21–36% of control values. Serum inhibin (as an index of FSH action) was suppressed in GnRH-A-treated animals by day 16, declining to 38% of control concentrations at day 25. Therefore, the primary lesion produced by GnRH-A induced gonadotrophin withdrawal is the rapid and profound reduction in the number of type B spermatogonia. The time course of germ cell loss suggests the inhibition of type A pale spermatogonial mitosis as the primary mechanism. Later germ cell maturation, specifically meiosis and spermiogenesis, appears to proceed unaffected. The decline in late spermatocytes and spermatids by 25 days of GnRH-A treatment is attributed to a 'depletional wave' from the spermatogonial lesion. The fact that such marked spermatogenic disruption occurs in the face of substantial testicular testosterone concentrations implies a significant role for FSH in spermatogonial development.
M Myers, K L Britt, N G M Wreford, F J P Ebling, and J B Kerr
Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 ± 286 (S.E.M.) and 2227 ± 101 primordial follicles, and 137 ± 25 and 265 ± 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.