TGF-β superfamily members including bone morphogenetic proteins (BMPs) and their receptors (BMPR-1A, -1B and -2) have been shown to be important for reproductive function in both males and females, while information on the role of BMPs in males is limited. Functional studies on select BMPs and BMP receptors have demonstrated vital roles for these proteins in somatic and germ cell proliferation, steroidogenesis and overall fertility. In order to gain insight into the importance of these genes during postnatal reproductive development in males, our study was undertaken to specify the distribution of BMP and BMPR mRNA in male reproductive and steroidogenic tissues and quantify these genes in the testis using the mouse as our model. We screened testis at two, four, six and eight weeks of age for the expression of ten BMPs and three BMP receptors using RT-qPCR. All three BMP receptor mRNAs – Bmpr1a, Bmpr1b and Bmpr2, and ten BMP mRNAs – Bmp2, Bmp3, Bmp3b, Bmp4, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b and Bmp15 were expressed in mouse testis at all stages screened. Testicular expression of genes varied within age groups and at specific developmental stages. Our study establishes an extensive BMP system in mouse reproductive and steroidogenic tissues.

The hormone leptin, which is thought to be primarily produced by adipose tissue, is a polypeptide that was initially characterized by its ability to regulate food intake and energy metabolism. Leptin appears to signal the status of body energy stores to the brain, resulting in the regulation of food intake and whole-body energy expenditure. Subsequently, it was recognized as a cytokine with a wide range of peripheral actions and is involved in the regulation of a number of physiological systems including reproduction. In the fed state, leptin circulates in the plasma in proportion to body adiposity in all species studied to date. However other factors such as sex, age, body mass index (BMI), sex steroids and pregnancy may also affect leptin levels in plasma. In pregnant mice and humans, the placenta is also a major site of leptin expression. Leptin circulates in biological fluids both as free protein and in a form that is bound to the soluble isoform of its receptor or other binding proteins such as one of the immunoglobulin superfamily members Siglec-6 (OB-BP1). Although the actions of leptin in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal axis, there have also been reports of local direct effects of leptin at the peripheral level, however, these data appear contradictory. Therefore, there is a need to summarize the current status of research outcomes and analyze the possible reasons for differing results and thus provide researchers with new insight in designing experiments to investigate leptin effect on reproduction. Most importantly, our recent experimental data suggesting that reproductive performance is improved by decreasing concentrations of peripheral leptin was unexpected and cannot be explained by hypotheses drawn from the experiments of excessive exogenous leptin administration to normal animals or ob/ob mice.

It is well documented that there is considerable batch to batch variation in the activity of commercial preparations of gonadotrophins. These products are used in a variety of assisted reproductive procedures in the livestock industry, consequently this high degree of variation between products and batches adds to the already considerable between animal variations in response to the treatment. Equine chorionic gonadotrophin (eCG) is a heterodimeric glycoprotein hormone secreted by the placental endometrial cups during the first third of gestation in the horse. Plasma is harvested from pregnant mares between 40–90 days of gestation and the eCG isolated and used to formulate commercial preparations. Previous research has shown that eCG like the other gonadotrophins is a highly heterogeneous molecule with significant differences in bioactivity between isoforms. The aim of this study was to determine whether significant differences in isoform composition exist between various commercial preparations of eCG (n = 15), and how this compares with the isoform composition found in plasma (n = 23). Concentrations of eCG were determined using a competitive eCG ELISA. Liquid phase iso-electric focusing was used to fractionate plasma and the commercial preparations into 10 pH ranges from pH 3.0 to pH 10.0. Data from the 10 fractions were grouped into acidic (pH 3.0–5.1), intermediate (pH 5.2–7.9), or basic (pH 8.0–10.0) isoform categories for analysis. Immunoactivity between commercial eCG products ranged from 44% to 362% of stated bioactivity. Iso-electric focusing showed that the majority of the immunoactivity (92%) of the commercial preparations was found in the acidic fractions (pH 3.0–5.1), and in particular in the pH range 3.0–3.8. This contrasted starkly with isoform profiles found in pregnant mare plasma samples which showed a much greater spread across all 3 pH ranges. In summary, the isolation processes of commercial eCG preparations appears to selectively favour the acidic isoforms of eCG.

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein hormone that is harvested from the plasma of pregnant mares for the formulation of commercial products which are used in a variety of assisted reproductive procedures in livestock. It is well documented that the bioactivity of gonadotrophin products is highly variable. The aim of this study was therefore to determine how different eCG products affected target tissues and cells. Isoforms of eCG were separated with iso-electric focusing and immunoactivity measured with RIA. For in vivo bioassay, dose-response eCG treatments were administered subcutaneously to immature female mice with follow-up treatment on day 2. On day 3 the mice were asphyxiated and ovaries dissected and weighed relative to standard. For in vitro bioassay, adult male mice were asphyxiated, testes removed, decapsulated, dispersed, strained and washed with DMEM:F12 0.1% BSA. The cell stock was counted and diluted for culture at 20,000 cells/well. Cell viability was determined using trypan blue. Five doses were tested for each eCG product. The cells were incubated at 32ºC for 3 hours in 5.0% CO2 in humidified conditions. Media was collected after 3 hours and immediately assayed for testosterone with RIA. Product eCG with ~90% isoforms with pH 3.0–3.7 and ~10% with pH 3.8–4.4, showed 29% greater biologically activity in the ovarian augmentation assay and 44.8% more immunoactivity then stated product bioactivity. Product eCG with ~70% isoforms with pH 3.0–3.7 and ~30% with pH 3.8–7.4 was 3.0% less effective in vivo and had 7.9% less immunoactivity then stated bioactivity, however, in vitro testosterone production was more effectively stimulated then with the previous more acidic eCG product. Our study shows a selective difference in commercial eCG biological activity that appears to depend on subtle isoform heterogeneity. Future studies aim to determine the specific actions of eCG isoforms in target tissues.

Bone morphogenetic proteins (BMPs) and receptors (BMPR-1A, BMPR-1B, BMPR-2) have been shown to be vital for female reproduction, while their roles in males are poorly described. Our study was undertaken to specify the function of BMPR-1B in steroidogenic enzyme gene expression, testosterone production and reproductive development in male mice, given that Bmpr1b mRNA is expressed in mouse testis and Bmpr1b knockout results in compromised fertility. Male mice were passively immunized for 6 days with anti-BMPR-1B in the presence or absence of exogenous gonadotrophins. We then measured the effects of anti-BMPR-1B on testicular hydroxysteroid dehydrogenase isoforms (Hsd3b1, Hsd3b6, and Hsd17b3) and aromatase (Cyp19) mRNA expression, testicular and serum testosterone levels, and testis and seminal vesicle weight. In vitro testosterone production in response to anti-BMPR-1B was determined using testicular culture, and Leydig cell culture in the presence or absence of gonadotrophins. In Leydig cell culture the contribution of seminiferous tubules and Leydig cells were examined by preconditioning the media with these testicular constituents. In adult mice, anti-BMPR-1B increased testosterone and Hsd3b1 but decreased Hsd3b6 and Cyp19 mRNA. In adult testicular culture and seminiferous tubule conditioned Leydig cell culture, anti-BMPR-1B reduced testosterone, while in normal and Leydig cell conditioned Leydig cell culture it increased testosterone levels. In pubertal mice, anti-BMPR-1B reduced gonadotrophin stimulated seminal vesicle growth. In conclusion, BMPR-1B has specific developmental functions in the autocrine and paracrine regulation of testicular steroidogenic enzyme gene expression and testosterone production in adults and in the development of seminal vesicles during puberty.