Increasing testicular temperature by exposure to elevated ambient temperatures restores spermatogenesis in adult Utp14b (jsd) mutant (jsd) mice

Abstract

Because mutations in the human UTP14C gene are associated with male infertility, we sought to develop a method for fertility restoration in azoospermic mice with a mutation in the orthologous Utp14b(jsd) (jsd) gene that have spermatogonial arrest. The method is based on our observation that elevation of testicular temperatures restores spermatogonial differentiation in jsd mutant mice. To non-surgically raise intrascrotal temperatures we placed these mice in incubators at different elevated ambient temperatures. Exposure of jsd/jsd mice to ambient temperatures of 34.5 °C or 35.5 °C for 24 days increased the proportion of tubules with spermatocytes from 0% in untreated controls to over 80%. As those higher temperatures interfere with spermatid differentiation, the mice were then transferred to incubators at 32-32.5 °C for the next 24 days. These environments allowed differentiation to progress, resulting in up to 42% of tubules having late spermatids and about half of the mutant mice having spermatozoa in testicular suspensions. When these spermatozoa were used in intracytoplasmic sperm injection, all gave rise to viable healthy offspring with normal weight gain and fertility. The successful restoration of fertility in Utp14b mutant mice suggests that transient testicular warming might also be useful for spermatogenesis recovery in infertile men with UTP14C gene mutations.

Keywords: azoospermia; genetic disorders; infertility; intracytoplasmic sperm injection; spermatogenesis.

Figure 1

Testis weights, percentages of tubules with late spermatids, and epididymal sperm counts in heterozygous +/jsd mice on C3H and HB129 genetic backgrounds after the induction period (A,B) and after the progression period (C-E). Numbers of mice evaluated per treatment group are indicated in parentheses. nd = not done. Data are means ± SEM; the sperm counts were averaged using log-transformed data. * Significantly different from mice maintained at room temperature (P < 0.05). “35.5”°C indicates mice were exposed to temperatures >36°C for 6-14 days as follows: # All 3 of the C3H-background mice evaluated at the end of the induction period; § 6 of the 7 C3H background mice evaluated at the end of the progression period. The slight but significant increase in testis weight in the HB129 mice initially exposed to 34°C seems anomalous.

Figure 2

Testicular histology (A-F) and sperm morphology (G-M). (A) +/jsd mouse at room temperature showing normal spermatogenesis and numerous late spermatids (ls). (B) +/jsd mice after incubation at 35.5°C showing round spermatids (rs) as the latest stage, and (C) after further incubation at 32.5°C showing the development of late spermatids is restored. (D) jsd/jsd mutant mouse at room temperature showing interphase (g) and mitotic (m) spermatogonia as the only germ cells. (E) jsd/jsd mouse after incubation at 35.5°C showing development of germ cells to the pachytene (p) spermatocyte stage, and (F) after further incubation at 32.5°C showing development to the late spermatid stages but fewer pachytene cells than at the end of the induction period; inset in F shows normal appearing late spermatid nuclei. (G) Sperm from testis of a +/jsd mouse. Sperm from testes of incubated (H) C3H-background jsd/jsd mutants, all of which were abnormal, and from HB129 jsd/jsd mutants, showing normal (I) and abnormal sperm (J). Cauda epididymal sperm from a wild-type male (K) and from a HB129-background jsd/jsd mutant showing normal (L) and abnormal sperm (M). Bars = 50 μm (A-F) and 20 μm (G-M).

Figure 3

Testis weights and percentages of tubules with spermatocytes and with late spermatids in jsd mutant mice on the C3H and HB129 genetic backgrounds after the induction period (A-C) and after the progression period (D-H). (A-E) Evaluation of spermatogenesis after different induction and progression temperatures. Numbers of mice per treatment group are indicated in parentheses. Numbers along abscissa indicate low values for which the bar is not visible. nd = not done. * Significantly different from mice maintained at room temperature (P < 0.05). “35.5”°C indicates mice were exposed to 35.5°C and also temperatures >36°C for 6-14 days as follows: # All 3 mice were exposed to >36°C for 20 days; § 6 of the 15 mice were exposed to >36°C for 8 to 14 days; ¶ 5 of the 10 mice were exposed to >36°C for 8 to 20 days. (G-H) Relationships between testis weights and the presence of late spermatids after induction/progression regimens of 34.5°/32°C or 35.5°/32.5°C for C3H-background (G) and HB129-background jsd/jsd mice (H). The correlation coefficients (r_s) and significance of the correlation (P) are given and regression lines are plotted. Mice from which testicular cell suspensions were prepared for ICSI but no sperm were found (□), and those from which sperm were found and ICSI was successful (◆). Mice not used for ICSI are indicated with (○).