Craig A. Harrison

Declining classroom attendance and in class engagement in Australian higher education could be a result of systemic factors and highlight the need for innovative teaching strategies. This study evaluated the impact of a flipped learning (FL) escape room workshop on student engagement and confidence in male reproductive physiology among second-year physiology students at Monash University. A total of 418 students (91% of the cohort) attended the non-compulsory workshop, themed around diagnosing the infertility of a fictional medieval king. Pre- and post-workshop surveys assessed engagement, preparedness, and confidence across five physiology topics. Of the attendees, 333 completed the pre-workshop and 368 completed the post-workshop surveys. Students reported high engagement with the workshop activities (mean = 90%), with significant differences in confidence based on pre-workshop preparedness. Post workshop confidence improved significantly across all topics, with the largest gains observed in the least prepared students. For example, students with 0% pre-workshop module completion reported confidence increases of 55–118% across the topics covered in the workshop, while those fully prepared showed 8–19% gains. These findings support the use of FL combined with gamified, narrative-driven workshops to enhance student engagement and confidence, regardless of their level of preparedness. The escape room format provided an inclusive environment that encouraged collaboration and active learning, demonstrating its potential as an engaging model for physiology education.

For female domestic cats, ovariohysterectomy is the only method of inducing permanent infertility. However, hundreds-of-millions of free-roaming cats globally highlight the necessity for alternative contraceptive approaches. One strategy involves a single injection of vector delivering a fertility-inhibiting protein for lifetime contraception. Recent studies in mice and cats have identified anti-Müllerian hormone as an excellent candidate for this type of contraception. Here, we leverage our recent characterization of the molecular mechanisms underlying human anti-Müllerian hormone synthesis and activity, to generate potent feline anti-Müllerian hormone analogs. Single intramuscular delivery of these analogs to female cats using an adeno-associated viral vector leads to a greater than 1000-fold increase in feline anti-Müllerian hormone levels, which are sustained for 9 months. High serum anti-Müllerian hormone is associated with abnormal estrus cyclicity, non-follicular ovarian cyst formation, and a progressive decline in antral follicle numbers, however, the few surviving large follicles continue to ovulate. Unlike previous studies, supraphysiologic levels of anti-Müllerian hormone do not block conception, although they are incompatible with the maintenance of pregnancy. Our findings highlight the complexity of the effects of anti-Müllerian hormone on ovarian physiology but confirm that this growth factor is a candidate for fertility control in free-roaming cats.

The roles of anti‐Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic–pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor‐binding interface, we generated a series of human AMH variants and assessed bioactivity. AMH _SCUT potency (EC ₅₀ 4 ng/mL) was increased 5‐ or 10‐fold by incorporating Gln ⁴⁸⁴ Met/Leu ⁵³⁵ Thr (EC ₅₀ 0.8 ng/mL) or Gln ⁴⁸⁴ Met/Gly ⁵³³ Ser (EC ₅₀ 0.4 ng/mL) mutations, respectively. Furthermore, the Gln ⁴⁸⁴ Met/Leu ⁵³⁵ Thr double mutant displayed enhanced efficacy, relative to AMH _SCUT . Finally, we identified residues within the wrist pre‐helix of AMH (Trp ⁴⁹⁴ , Gln ⁴⁹⁶ , Ser ⁴⁹⁷ , and Asp ⁴⁹⁸ ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain‐ (Trp ⁴⁹⁴ Phe or Gln ⁴⁹⁶ Leu) or loss‐ (Ser ⁴⁹⁷ Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step‐wise receptor‐complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Inhibins are members of the transforming growth factor-β family, composed of a common α-subunit disulfide-linked to 1 of 2 β-subunits (βA in inhibin A or βB in inhibin B). Gonadal-derived inhibin A and B act in an endocrine manner to suppress the synthesis of follicle-stimulating hormone (FSH) by pituitary gonadotrope cells. Roles for inhibins beyond the pituitary, however, have proven difficult to delineate because deletion of the inhibin α-subunit gene (Inha) results in unconstrained expression of activin A and activin B (homodimers of inhibin β-subunits), which contribute to gonadal tumorigenesis and lethal cachectic wasting. Here, we generated mice with a single point mutation (Arg²³³Ala) in Inha that prevents proteolytic processing and the formation of bioactive inhibin. In vitro, this mutation blocked inhibin maturation and bioactivity, without perturbing activin production. Serum FSH levels were elevated 2- to 3-fold in Inha^R233A/R233A mice due to the loss of negative feedback from inhibins, but no pathological increase in circulating activins was observed. While inactivation of inhibin A and B had no discernible effect on male reproduction, female Inha^R233A/R233A mice had increased FSH-dependent follicle development and enhanced natural ovulation rates. Nevertheless, inhibin inactivation resulted in significant embryo-fetal resorptions and severe subfertility and was associated with disrupted maternal ovarian function. Intriguingly, heterozygous Inha^+/R233A females had significantly enhanced fecundity, relative to wild-type littermates. These studies have revealed novel effects of inhibins in the establishment and maintenance of pregnancy and demonstrated that partial inactivation of inhibin A/B is an attractive approach for enhancing female fertility.

Discovery of a novel pituitary protein suggests a new means to enhance fertility.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-β (TGF-β1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models of disease.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid‐derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT‐PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix‐related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Oocyte-secreted factors, bone morphogenetic protein-15 (BMP15) and growth differentiation factor-9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n=154), and from peri- and post-menopausal women (n=28). Serum concentrations of BMP15 and GDF9 in women relative to age, AMH, number of oocytes retrieved, and polycystic ovaries/syndrome (PCO(S)) after superovulation for IVF. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/ml, respectively) and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64- and 15-fold respectively between women, but did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15, was associated with oocyte number retrieved in non-PCO(S) patients (p=0.018). GDF9 and BMP15 associations with oocyte number differed significantly (pp=0.020). Serum BMP15, but not GDF9, was lower in women over 55 years, compared to women of reproductive age (p<0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum, and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.