Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5 mm), pre-ovulatory follicles (6.0-7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.

Although much progress has been made in the genetic dissection of biological networks involved in follicular/luteal development in the mammalian ovary, the gene regulation mechanisms involved are still poorly understood. Over the last 10 years, miRNAs have emerged as master regulators of tissue growth and differentiation in animals. However, compared with other body tissues, little is still known about the functional involvement of miRNAs in the ovary. Several studies have identified miRNA populations specifically associated with the development of follicles and corpora lutea, particularly in relation to the follicular-luteal transition, and the functional involvement of some of these miRNAs has been characterised in vitro and/or in vivo. Specifically, three different miRNAs, miR-224, miR-378 and miR-383, have shown to be involved in regulating aromatase expression during follicle development. In addition, miR-21 has been identified as promoting follicular cell survival during ovulation, and pro-angiogenic miR-17-5p and let-7b were shown to be necessary for normal development of the corpus luteum. Experimental evidence for the involvement of several other miRNAs in different aspects of follicle/luteal development has also been obtained. In addition, many of these studies exemplify the challenges associated with identifying physiologically relevant targets of ovarian miRNAs. Continuous advances in this field will be considerably facilitated by progress in understanding miRNA physiology in other body systems and will eventually lead to a much better understanding of the control of follicular/luteal development. In turn, through the potential offered by miRNA diagnostics and miRNA therapeutics, this new knowledge should bring considerable benefits to reproductive medicine.

Several different miRNAs have been proposed to regulate ovarian follicle function; however, very limited information exists on the spatiotemporal patterns of miRNA expression during follicle development. The objective of this study was to identify, using microarray, miRNA profiles associated with growth and regression of dominant-size follicles in the bovine monovular ovary and to characterize their spatiotemporal distribution during development. The follicles were collected from abattoir ovaries and classified as small (4-8 mm) or large (12-17 mm); the latter were further classified as healthy or atretic based on estradiol and CYP19A1 levels. Six pools of small follicles and individual large healthy (nZ6) and large atretic (nZ5) follicles were analyzed using Exiqon's miRCURY LNA microRNA Array 6th gen, followed by qPCR validation. A total of 17 and 57 sequences were differentially expressed (greater than or equal to twofold; P!0.05) between large healthy and each of small and large atretic follicles respectively. Bovine miRNAs confirmed to be upregulated in large healthy follicles relative to small follicles (bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873) were further characterized. Three of these miRNAs (bta-miR-144, bta-miR-202, and bta-miR-873) were also downregulated in large atretic follicles relative to large healthy follicles. Within the follicle, these miRNAs were predominantly expressed in mural granulosa cells. Further, body-wide screening revealed that bta-miR-202, but not other miRNAs, was expressed exclusively in the gonads. Finally, a total of 1359 predicted targets of the five miRNAs enriched in large healthy follicles were identified, which mapped to signaling pathways involved in follicular cell proliferation, steroidogenesis, prevention of premature luteinization, and oocyte maturation.

Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (nZ6 mares). Follicular fluid levels of progesterone and estradiol were lower (P!0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P!0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P%0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (nZ5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (PZ0.05) and lower granulosa cell levels of CYP19A1 and LHCGR (P!0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P!0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets, PTEN, RASA1, and SMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

The present paper describes, to our knowledge for the first time, the successful collection and evaluation of semen from the Indian white-backed vulture (Gyps bengalensis), a critically endangered bird. Over a period of 2 yr, semen was collected using the manual massage method and evaluated for semen volume, semen pH, sperm concentration, percentage normal/abnormal spermatozoa, and percentage motile spermatozoa. It appears that the concentration of spermatozoa and percentage motile spermatozoa in the Indian white-backed vultures are low compared to those in other birds. Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) proved to be the best semen extender compared to two others (Beltsville Poultry Semen Extender and Lake diluent). Furthermore, TALP with 20% egg yolk and supplemented with 8% dimethyl sulfoxide maintained 50% of the initial percentage of motile spermatozoa following cryopreservation and thawing. A computer-aided semen analysis indicated that the spermatozoa of the Indian white-backed vulture are extremely active and swim in linear trajectories for up to 5 h following dilution in TALP. The trajectories were linear with time, but we noticed a decrease in the velocity parameters (average path velocity, curvilinear velocity, and progressive velocity). Thus, the present study provides baseline data on semen characteristics of the highly endangered Indian white-backed vulture, and these data could be of immense importance to reproductive and conservation biologists attempting to breed these animals in captivity, which to date has not been achieved.