Synergistic enhancement of the mouse Pramex1 and Pramel1 in repressing retinoic acid (RA) signaling during gametogenesis.

BACKGROUND: PRAME constitutes one of the largest multi-copy gene families in Eutherians, encoding cancer-testis antigens (CTAs) with leucine-rich repeats (LRR) domains, highly expressed in cancer cells and gametogenic germ cells. This study aims to elucidate genetic interactions between two members, Pramex1 and Pramel1, in the mouse Prame family during gametogenesis using a gene knockout approach. RESULT: Single-gene knockout (sKO) of either Pramex1 or Pramel1 resulted in approximately 7% of abnormal seminiferous tubules, characterized by a Sertoli-cell only (SCO) phenotype, impacting sperm count and fecundity significantly. Remarkably, sKO female mice displayed normal reproductive functions. In contrast, Pramex1/Pramel1 double knockout (dKO) mice exhibited reduced fecundity in both sexes. In dKO females, ovarian primary follicle count decreased by 50% compared to sKO and WT mice, correlating with a 50% fecundity decrease. This suggested compensatory roles during oogenesis in Pramex1 or Pramel1 sKO females. Conversely, dKO males showed an 18% frequency of SCO tubules, increased apoptotic germ cells, and decreased undifferentiated spermatogonia compared to sKO and WT testes. Western blot analysis with PRAMEX1- or PRAMEL1-specific antibodies on sKO testes revealed compensatory upregulation of each protein (30-50%) in response to the other gene's deletion. Double KO males exhibited more severe defects in sperm count and litter size, surpassing Pramex1 and Pramel1 sKO accumulative effects, indicating a synergistic enhancement interaction during spermatogenesis. Additional experiments administering trans-retinoic acid (RA) and its inhibitor (WIN18,446) in sKO, dKO, and WT mice suggested that PRAMEX1 and PRAMEL1 synergistically repress the RA signaling pathway during spermatogenesis. CONCLUSION: Data from sKO and dKO mice unveil a synergistic interaction via the RA signaling pathway between Pramex1 and Pramel1 genes during gametogenesis. This discovery sets the stage for investigating interactions among other members within the Prame family, advancing our understanding of multi-copy gene families involved in germ cell formation and function.

Antral follicle counts and association with ovarian superstimulatory response to gonadotropins in prepubertal calves.

Antral follicle count (AFC) repeatability at the time of follicular wave emergence and duration of gonadotropin treatment in calves with small and large AFC affects the superstimulatory response of follicles. In Study I, the individual AFC was determined, calves were ranked as having a small, medium or large AFC, and a second count was performed prior to FSH treatments. There was a positive association between the number of follicles ≥1 mm after the first and second counts (r = 0.4; P = 0.003). In Study II, calves with small and large AFC were administered pFSH for 4 or 7 days, pLH 20 h after last pFSH administration and cumulus-oocyte-complexes (COC) were collected. In calves having large as compared with small AFC, number of follicles ≥6 mm were greater (P = 0.01) and COC collected were greater (P = 0.001). The proportion of large-sized follicles (>9 mm) was greater in the 7-day than in the 4-day gonadotropin treatment group (56.4 ±â€¯8.3 and27.8 ±â€¯7.5 %, respectively; P = 0.01). In Study III, there was a positive association between AFC and number of follicles ≥6 mm at the time of COC collection (r = 0.6; P = 0.003). In summary, the number of follicles at the time of follicular wave emergence was associated with the number of follicles recruited during subsequent waves of follicular development and ovarian response following gonadotropin superstimulation. Calves with a large AFC had more COC collected than calves with a small AFC.

Effect of dose and duration of FSH treatment on ovarian response in prepubertal calves.

The objective of the study was to determine relative effects of dose (200 or 350 mg) and duration (4 or 7 days) of superstimulatory treatment on the ovarian response in prepubertal calves. Calves with similar antral follicular counts at wave emergence (n = 24) were given eight doses of either 25 or 44 mg pFSH every 12 h for 4 days or 14 doses of either 14 or 25 mg pFSH for 7 days beginning at the time of follicular wave emergence and 12.5 mg of pLH im 12 h after the last FSH treatment. On Day 4 of pFSH treatment, calves given 14 mg had fewer follicles ≥3 mm than those given 25 mg (15.1 ± 1.9 and 27.9 ± 3.3, respectively; P = 0.04). At the end of treatment (24 h post-LH), number of follicles ≥9 mm was greater in calves of groups treated with 350 than 200 mg (13.5 ± 1.8 and 8.8 ± 1.3, respectively; P = 0.02) and calves of groups treated for 7 than 4 days (13.3 ± 1.8 and 9.0 ± 1.3, respectively; P = 0.03). The number of spontaneous ovulations was greater in calves of groups treated for 7 than 4 days as was the total number of ovulations (9.7 ± 0.9 and 6.9 ± 1.0, respectively; P ≤ 0.05). In summary, a dose of 25 mg of pFSH per treatment given twice daily for 7 days resulted in a greater ovarian response than other superstimulatory treatments in prepubertal calves.

Associations between resumption of postpartum ovarian activity, uterine health and concentrations of metabolites and acute phase proteins during the transition period in Holstein cows.

The resumption of ovarian activity, uterine health, severity of the negative energy balance and the synthesis of inflammatory mediators during the transition period in dairy cows are interrelated. Therefore, the aim of this study was to evaluate the association between the resumption of postpartum ovarian activity and the percentage of polymorphonuclear (PMN) cells in endometrial cytology, lipid mobilization and the secretion of acute phase proteins. For this study, 20 multiparous Holstein cows were used. Blood samples that were collected from 21d before calving to 44d in milk (DIM) were analyzed for serum glucose, non-esterified fatty acids (NEFA), insulin, haptoglobin, albumin, paraoxonase and progesterone. Endometrial cytology was performed at 37±2DIM to evaluate the percentage of PMN cells in the uterine flushing. Cows were divided into two groups: (1) ovulatory cows (n=12), which returned to ovarian activity by 44±2DIM; and (2) anovulatory cows (n=8), which did not resume ovarian activity during this period. Ovulatory cows had a lower (P=0.05) percentage of PMN cells in endometrial cytology than anovulatory cows (26.3±8.3% vs. 53.4±16.9%, respectively). Ovulatory cows had higher serum albumin during the pre- (P=0.03) and postpartum periods (P=0.01), and tended to have lower haptoglobin concentrations in the prepartum period (P=0.07) and higher paraoxonase activity in the postpartum period (P=0.09). In conclusion, cows that resumed ovarian activity early in the postpartum period had higher albumin concentrations in the peripartum period, which were associated with a lower percentage of uterine PMN cells.