Monitoring sexual steroids and cortisol at different stages of the ovarian cycle from two capuchin monkey species: use of non- or less invasive methods than blood sampling.

Endocrine monitoring of non-human primates (NHP) via faecal metabolites of steroid hormones appears as a useful non-invasive alternative to evaluate the reproductive status of free living NHP, as well as of those kept in captivity but of difficult handling. However, validation is needed with plasma values before its application in the field. The aim of the present study was to monitor the different phases of the menstrual cycle from the new world NHP Sapajus apella and S. libidinosus. For this, hormonal and faecal plasma levels of E2, P4 and cortisol were assessed during different days of the menstrual cycle, together with colpocitology. The mean duration of the menstrual cycle according colpocitology was of 21.7 and 21.0 days for S. apella and S. libidinosus, respectively. These values were similar to those observed via plasma analysis, i.e. 22.7 and 20.3 days for S. apella and S. libidinosus, respectively. The day of plasmatic E2 peak was set as Day -1 and the estimated day of ovulation was set as Day 0 and occurred two days earlier in S. libidinosus than in S. apella females. In both species, it was observed a delay in faecal E2 peak of six days for S. apella and of 11 days for S. libidinosus when compared with the plasma peak. A maximum P4 plasma concentration was observed in the middle of luteal phase in S. apella and in S. libidinosus, both at around day 5. However, faecal P4 peaks were detected at days 9 and 8 in S. apella and S. libidinosus, respectively. Mean plasma and faecal cortisol levels were variable during all ovulatory cycle of S. apella and S. libidinosus females. Although no exact correlation was observed between plasmatic and faecal profile of steroid hormone, faecal samples were able to indicate ovarian cycle phase, being important to assess the reproductive status of the females applying a non-invasive method.

Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi).

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella).

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.