Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF(1) rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.

Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.

Suppression of intratesticular testosterone (ITT) levels is required for spermatogenic recovery in rats after irradiation, but maintenance of peripheral testosterone (T) levels is important for many male functions. Considering the preservation of peripheral T while suppressing ITT, we tested the effects of a combination of a progestin, medroxyprogesterone acetate (MPA), plus T on spermatogenic recovery after irradiation, and compared its effects to those of T alone or T combined with estradiol (E2). Rats were given testicular irradiation (6 Gy) and treated during wk 3-7 after irradiation with MPA + T, or the individual steroids with or without GnRH antagonist (GnRH-ant), or GnRH-ant alone, or T + E2. Whereas GnRH-ant alone stimulated differentiation in 55% of tubules 13 wk after irradiation compared with 0% in irradiated-only rats, the addition of MPA reduced the percentage of tubules showing differentiation to 18%. However, T or MPA alone or the combination of the two induced germ cell differentiation in only 2-4% of tubules. In contrast, E2 stimulated differentiation in 88% of tubules, and T combined with E2 still resulted in differentiation in 30% of tubules. Although both MPA and E2 suppressed ITT levels to approximately 2% of control (2 ng/g testis), MPA was a less effective stimulator of spermatogenic recovery than E2 or GnRH-ant alone. MPA's function as a weak androgen was likely responsible for inhibiting spermatogenic recovery, as was the case for all other tested androgens. Thus, for clinical protection or restoration of spermatogenesis after radiation or chemotherapy by suppressing T production, MPA, at least in the doses used in the present study, is suboptimal. The combination of an estrogen with T appears to be most effective for stimulating such recovery.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting.MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility.MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility. K E Y W O R D S male fertility, microRNA, real-time quantitative RT-PCR, sperm How to cite this article: Menezes ESB, Badial PR, El Debaky H, et al. Sperm miR-15a and miR-29b are associated with bull fertility. Andrologia. 2020;52:e13412. https ://doi.