Division Events Research Articles

Deep learning for rapid analysis of cell divisions in vivo during epithelial morphogenesis and repair.

Cell division is fundamental to all healthy tissue growth, as well as being rate-limiting in the tissue repair response to wounding and during cancer progression. However, the role that cell divisions play in tissue growth is a collective one, requiring the integration of many individual cell division events. It is particularly difficult to accurately detect and quantify multiple features of large numbers of cell divisions (including their spatio-temporal synchronicity and orientation) over extended periods of time. It would thus be advantageous to perform such analyses in an automated fashion, which can naturally be enabled using deep learning. Hence, we develop a pipeline of deep learning models that accurately identify dividing cells in time-lapse movies of epithelial tissues in vivo. Our pipeline also determines their axis of division orientation, as well as their shape changes before and after division. This strategy enables us to analyse the dynamic profile of cell divisions within the Drosophila pupal wing epithelium, both as it undergoes developmental morphogenesis and as it repairs following laser wounding. We show that the division axis is biased according to lines of tissue tension and that wounding triggers a synchronised (but not oriented) burst of cell divisions back from the leading edge.

Deep learning for rapid analysis of cell divisions in vivo during epithelial morphogenesis and repair

Cell division is fundamental to all healthy tissue growth, as well as being rate-limiting in the tissue repair response to wounding and during cancer progression. However, the role that cell divisions play in tissue growth is a collective one, requiring the integration of many individual cell division events. It is particularly difficult to accurately detect and quantify multiple features of large numbers of cell divisions (including their spatio-temporal synchronicity and orientation) over extended periods of time. It would thus be advantageous to perform such analyses in an automated fashion, which can naturally be enabled using deep learning. Hence, we develop a pipeline of deep learning models that accurately identify dividing cells in time-lapse movies of epithelial tissues in vivo. Our pipeline also determines their axis of division orientation, as well as their shape changes before and after division. This strategy enables us to analyse the dynamic profile of cell divisions within the Drosophila pupal wing epithelium, both as it undergoes developmental morphogenesis and as it repairs following laser wounding. We show that the division axis is biased according to lines of tissue tension and that wounding triggers a synchronised (but not oriented) burst of cell divisions back from the leading edge.

Mechanistic modeling of cell viability assays with in silico lineage tracing.

Data from cell viability assays, which measure cumulative division and death events in a population and reflect substantial cellular heterogeneity, are widely available. However, interpreting such data with mechanistic computational models is hindered because direct model/data comparison is often muddled. We developed an algorithm that tracks simulated division and death events in mechanistically detailed single-cell lineages to enable such a model/data comparison and suggest causes of cell-cell drug response variability. Using our previously developed model of mammalian single-cell proliferation and death signaling, we simulated drug dose response experiments for four targeted anti-cancer drugs (alpelisib, neratinib, trametinib and palbociclib) and compared them to experimental data. Simulations are consistent with data for strong growth inhibition by trametinib (MEK inhibitor) and overall lack of efficacy for alpelisib (PI-3K inhibitor), but are inconsistent with data for palbociclib (CDK4/6 inhibitor) and neratinib (EGFR inhibitor). Model/data inconsistencies suggest (i) the importance of CDK4/6 for driving the cell cycle may be overestimated, and (ii) that the cellular balance between basal (tonic) and ligand-induced signaling is a critical determinant of receptor inhibitor response. Simulations show subpopulations of rapidly and slowly dividing cells in both control and drug-treated conditions. Variations in mother cells prior to drug treatment all impinging on ERK pathway activity are associated with the rapidly dividing phenotype and trametinib resistance. This work lays a foundation for the application of mechanistic modeling to large-scale cell viability assay datasets and better understanding determinants of cellular heterogeneity in drug response.

The combination of sitagliptin and bee honey extract potentiates the anti-proliferative properties of 5-fluorouracil on Caco-2 cell line without detectable inflammatory events

BackgroundColon cancer originates in the colon, specifically the large intestine. It carries a poor prognosis and high mortality rate due to late diagnosis and migration. ObjectiveHere our objective was to evaluate the anticancer effects of sitagliptin (Sita) in colon cancer using Caco-2 cells. Additionally, we examined the role of bee honey extract in modulating cancer cell division and necrotic events commonly observed during drug treatments. MethodsWe monitored cell viability rate to evaluate the effect of bee honey extract compared to 5-fluorouracil (5-Fu), Sita, and their combinations. To gain further foresights into the implicated molecular interaction, we assessed the expression outline of Raf-1 and MEK, as proliferation effectors. Additionally, we examined the expression outline of p53 and Caspase 3, which are associated with programmed cell death (PCD), through western blot analysis. ResultsWe identified the Raf-1 expression pattern as a likely target for the drug combination and bee honey extract (HE), which effectively controlled colon cancer cell proliferation. Our study demonstrates that honey extract, either alone or in combination with drugs, can induce PCD by restoring the p53 and CASP-3 proteins. This was accompanied by a synergistic effect on the production of apoptotic cytokines, particularly interlukine-6 (IL-6) and IL-8, in cancer cells. Moreover, the treatment modulated the levels of pro-inflammatory cytokines, including IL-1α and IL-1β and anti-inflammatory cytokines, including IL-4 and IL-10. ConclusionOur findings shed light on honey extract and its combinations with 5-Fu and Sita in stimulating PCD and modulating cytokine production in Caco-2 cells.

Growing bacterial colonies harness emergent genealogical demixing to regulate organizational entropy

Spatiotemporal organization of individuals within growing bacterial colonies is a key determinant of intraspecific interactions and colony-scale heterogeneities. The evolving cellular distribution, in relation to the genealogical lineage, is thus central to our understanding of bacterial fate across scales. Yet, how bacteria self-organize genealogically as a colony expands has remained unknown. Here, by developing a custom-built label-free algorithm, we track and study the genesis and evolution of emergent self-similar genealogical enclaves, whose dynamics are governed by biological activity. Topological defects at enclave boundaries tune finger-like morphologies of the active interfaces. The Shannon entropy of cell arrangements reduce over time; with faster-dividing cells possessing higher spatial affinity to genealogical relatives, at the cost of a well-mixed, entropically favorable state. Our coarse-grained lattice model demonstrates that genealogical enclaves emerge due to an interplay of division-mediated dispersal, stochasticity of division events, and cell-cell interactions. The study reports so-far hidden emergent self-organizing features arising due to entropic suppression, ultimately modulating intraspecific genealogical distances within bacterial colonies.

Direct single-cell observation of a key Escherichia coli cell-cycle oscillator.

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.

Stochastic journeys of cell progenies through compartments and the role of self-renewal, symmetric and asymmetric division

Division and differentiation events by which cell populations with specific functions are generated often take place as part of a developmental programme, which can be represented by a sequence of compartments. A compartment is the set of cells with common characteristics; sharing, for instance, a spatial location or a phenotype. Differentiation events are transitions from one compartment to the next. Cells may also die or divide. We consider three different types of division events: (i) where both daughter cells inherit the mother’s phenotype (self-renewal), (ii) where only one of the daughters changes phenotype (asymmetric division), and (iii) where both daughters change phenotype (symmetric division). The self-renewal probability in each compartment determines whether the progeny of a single cell, moving through the sequence of compartments, is finite or grows without bound. We analyse the progeny stochastic dynamics with probability generating functions. In the case of self-renewal, by following one of the daughters after any division event, we may construct lifelines containing only one cell at any time. We analyse the number of divisions along such lines, and the compartment where lines terminate with a death event. Analysis and numerical simulations are applied to a five-compartment model of the gradual differentiation of hematopoietic stem cells and to a model of thymocyte development: from pre-double positive to single positive (SP) cells with a bifurcation to either SP4 or SP8 in the last compartment of the sequence.

Isolated Random Forest Assisted Spatio-Temporal Ant Colony Evolutionary Algorithm for Cell Tracking in Time-Lapse Sequences.

Multi-Object tracking in real world environments is a tough problem, especially for cell morphogenesis with division. Most cell tracking methods are hard to achieve reliable mitosis detection, efficient inter-frame matching, and accurate state estimation simultaneously within a unified tracking framework. In this paper, we propose a novel unified framework that leverages a spatio-temporal ant colony evolutionary algorithm to track cells amidst mitosis under measurement uncertainty. Each Bernoulli ant colony representing a migrating cell is able to capture the occurrence of mitosis through the proposed Isolation Random Forest (IRF)-assisted temporal mitosis detection algorithm with the assumption that mitotic cells exhibit unique spatio-temporal features different from non-mitotic ones. Guided by prediction of a division event, multiple ant colonies evolve between consecutive frames according to an augmented assignment matrix solved by the extended Hungarian method. To handle dense cell populations, an efficient group partition between cells and measurements is exploited, which enables multiple assignment tasks to be executed in parallel with a reduction in matrix dimension. After inter-frame traversing, the ant colony transitions to a foraging stage in which it begins approximating the Bernoulli parameter to estimate cell state by iteratively updating its pheromone field. Experiments on multi-cell tracking in the presence of cell mitosis and morphological changes are conducted, and the results demonstrate that the proposed method outperforms state-of-the-art approaches, striking a balance between accuracy and computational efficiency.

Mechanical and biochemical feedback combine to generate complex contractile oscillations in cytokinesis

The actomyosin cortex is an active material that generates force to drive shape changes via cytoskeletal remodeling. Cytokinesis is the essential cell division event during which a cortical actomyosin ring closes to separate two daughter cells. Our active gel theory predicted that actomyosin systems controlled by a biochemical oscillator and experiencing mechanical strain would exhibit complex spatiotemporal behavior. To test whether active materials invivo exhibit spatiotemporally complex kinetics, we imaged the C.elegans embryo with unprecedented temporal resolution and discovered that sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Contractile oscillations exhibited a range of periodicities, including those much longer periods than the timescale of RhoA pulses, which was shorter in cytokinesis than in any other biological context. Modifying mechanical feedback invivo or in silico revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Fast local ring ingression occurs where speed oscillations have long periods, likely due to increased local stresses and, therefore, mechanical feedback. Fast ingression also occurs where material turnover is high, invivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and, therefore, makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows for sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Thus, like biochemical feedback, mechanical feedback affords active materials responsiveness and robustness.

Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

Dislike of general opinion makes for tight elections.

In modern democracies, the outcome of elections and referendums is often remarkably tight. The repetition of these divisive events are the hallmark of a split society; to the physicist, however, it is an astonishing feat for such large collections of diverse individuals. Many sociophysics models reproduce the emergence of collective human behavior with interacting agents, which respond to their environment according to simple rules, modulated by random fluctuations. A paragon of this class is the Ising model which, when interactions are strong, predicts that order can emerge from a chaotic initial state. In contrast with many elections, however, this model favors a strong majority. Here we introduce a new element to this classical theory, which accounts for the influence of opinion polls on the electorate. This brings about a new phase in which two groups divide the opinion equally. These political camps are spatially segregated, and the sharp boundary that separates them makes the system size dependent, even in the limit of a large electorate. Election data show that, since the early 1990s, countries with more than about a million voters often found themselves in this state, whereas elections in smaller countries yielded more consensual results. We suggest that this transition hinges on the electorate's awareness of the general opinion.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Distinct dynamics and proximity networks of hub proteins at the prey-invading cell pole in a predatory bacterium.

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar "hub" proteins. Whereas most described bacteria inherit an "old" pole from the mother cell and a "new" pole from cell division, generating cell asymmetry at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium Bdellovibrio bacteriovorus, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in B. bacteriovorus. RomR already marks the future anterior poles of the progeny during the predator's growth phase, during a precise period closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomR's stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behavior changes shortly before the division of the elongated predator cell, where DivIVA accumulates at all septa and both poles. In vivo protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and reinforce the importance of the anterior "invasive" cell pole in prey-predator interactions. Our work also emphasizes the precise spatiotemporal order of cellular processes underlying B. bacteriovorus proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division.IMPORTANCEIn bacteria, cell poles are crucial areas where "hub" proteins orchestrate lifecycle events through interactions with multiple partners at specific times. While most bacteria exhibit one "old" and one "new" pole, inherited from the previous division event, setting polar identity poses challenges in bacteria with non-binary division. This study explores polar proteins in the predatory bacterium Bdellovibrio bacteriovorus, which undergoes filamentous growth followed by non-binary division inside another bacterium. Our research reveals distinct localization dynamics of the polar proteins RomR and DivIVA, highlighting RomR as an early "hub" marking polar identity in the filamentous mother cell. Using miniTurbo-based proximity labeling, we uncovered their unique protein networks. Overall, our work provides new insights into the cell polarity in non-binary dividing bacteria.

FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

The microRNA7833-AUX6 module plays a critical role in wood development by modulating cellular auxin influx in Populus tomentosa.

The critical role of auxin on secondary vascular development in woody plants has been demonstrated. The concentration gradient of endogenous indole-3-acetic acid and the cellular and molecular pathways contributing to the auxin-directed vascular organization and wood growth have been uncovered in recent decades. However, our understanding of the roles and regulations of auxin influx in wood formation in trees remains limited. Here, we reported that a microRNA, miR7833, participates in the negative regulation of stem cambial cell division and secondary xylem development in Populus tomentosa. The miR7833 is mainly expressed in the vascular cambium during stem radical growth and specifically targets and represses two AUX/LAX family auxin influx carriers, AUX5 and AUX6, in poplar. We further revealed that poplar AUX6, the most abundant miR7833 target in the stem, is preferentially enriched in the developing xylem and is a positive regulator for cell division and differentiation events during wood formation. Moreover, inhibition of auxin influx carriers by 1-naphthoxyacetic acids abolished the regulatory effects of miR7833 and AUX6 on secondary xylem formation in poplar. Our results revealed the essential roles of the miR7833-AUX6 module in regulating cellular events in secondary xylem development and demonstrated an auxin influx-dependent mechanism for wood formation in poplar.

Label-free analysis of bacterial growth and lysis at the single-cell level using droplet microfluidics and object detection-oriented deep learning

Bacteria identification and counting at the small population scale is important to many applications in the food safety industry, the diagnostics of infectious diseases and the study and discovery of novel antimicrobial compounds. There is still a lack of easy to implement, fast and accurate methods to count populations of motile cells at the single-cell level. Here, we report a label-free method to count and localize bacterial cells freely swimming in microfluidic anchored picolitre droplets. We used the object detection oriented YOLOv4 deep learning framework for cell detection from bright-field images obtained with an automated Z-stack setup. The neural network was trained to recognize Escherichia coli cell morphology with an average precision of approximately 84%. This allowed us to accurately identify individual cell division events, enabling the study of stochastic bacterial growth starting from initial populations as low as one cell. This work also demonstrates the ability to study single cell lysis in the presence of T7 lytic bacterial viruses (phages). The high precision in cell numbers facilitated the visualization of bacteria-phage interactions over timescale of hours, paving the way towards deciphering phage life cycles in confined environments.

First-passage-time statistics of growing microbial populations carry an imprint of initial conditions

In exponential population growth, variability in the timing of individual division events and environmental factors (including stochastic inoculation) compound to produce variable growth trajectories. In several stochastic models of exponential growth we show power-law relationships that relate variability in the time required to reach a threshold population size to growth rate and inoculum size. Population-growth experiments in E. coli and S. aureus with inoculum sizes ranging between 1 and 100 are consistent with these relationships. We quantify how noise accumulates over time, finding that it encodes—and can be used to deduce—information about the early growth rate of a population.

Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria.

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like structure (Z-ring) in the middle of the cell that serves as a scaffold for other division proteins. Super-resolution microscopy in bacterial models Escherichia coli and Bacillus subtilis showed that the Z-ring is discontinuous, while live cell imaging studies demonstrated that FtsZ moves along the ring by a mechanism known as treadmilling. To study the dynamics of FtsZ in vivo, a special cell placement in a vertical position is necessary for imaging the complete structure of the ring in the XY plane. In the case of FtsZ imaging in multicellular cyanobacteria, such as Anabaena sp. PCC7120, maintaining the filaments in a vertical position is challenging because of the size of the cells and the filaments' length. In this article, we describe a method that allows the vertical immobilization of Anabaena sp. PCC 7120 filaments using low melting point agarose and syringes, to record the Z-ring in a mutant that expresses a FtsZ-sfGFP fusion protein. This method is a rapid and inexpensive way to register protein dynamics at the division site using confocal microscopy.

Plaque vicinity as a hotspot of microglial turnover in a mouse model of Alzheimer's disease.

Microglia, the major immune cells of the brain, are functionally heterogeneous but in vivo functional properties of these cells are rarely studied at single-cell resolution. By using microRNA-9 regulated viral vectors for multicolor labeling and longitudinal in vivo monitoring of individual microglia, we followed their fate in the cortex of healthy adult mice and at the onset of amyloidosis in a mouse model of Alzheimer's disease. In wild-type mice, microglia were rather mobile (16% of the cells migrated at least once in 10-20 days) but had a low turnover as documented by low division and death rates. Half of the migratory events were tightly associated with blood vessels. Surprisingly, basic migration properties of microglia (i.e., fraction of migrating cells, saltatory migration pattern, speed of migration, translocation distance, and strong association with blood vessels) were preserved in amyloid-depositing brains, despite amyloid plaques becoming the major destination of migration. Besides, amyloid deposition significantly increased microglial division and death rates. Moreover, the plaque vicinity became a hotspot of microglial turnover, harboring 33% of all migration, 70% of death and 54% of division events.

The effects of endogenous FSH and its receptor on oogenesis and folliculogenesis in female Alligator sinensis

BackgroundThe precise mechanisms of hormone action responsible for the full course of events modulating folliculogenesis in crocodilian have not been determined, although histological features have been identified.ResultsThe Alligator sinensis ovarian morphological characteristics observed at 1, 15, 30, 60, 90, and 300 days post hatching(dph) revealed that the dynamic changes in germ cells varied in different meiotic and developmental stages, confirming that the processes of folliculogenesis were protracted and asynchronous. The presence of endogenous follicle-stimulating hormone(FSH) mRNA and protein expression within the cerebrum at 1 dph, in parallel with the increase in germ cells within the germ cell nests(Nest) from 1 dph to 15 dph, suggested that endocrine regulation of the pituitary-gonad axis is an early event in oogonia division. Furthermore, the endogenous expression of FSH showed a trend of negative feedback augmentation accompanied by the exhaustion of maternal yolk E2 observed at 15 dph. Such significant elevation of endogenous FSH levels was observed to be related to pivotal events in the transition from mitosis to meiosis, as reflected by the proportion of oogonia during premeiosis interphase, with endogenous FSH levels reaching a peak at the earliest time step of 1 dph. In addition, the simultaneous upregulation of premeiotic marker STRA8 mRNA expression and the increase in endogenous FSH further verified the above speculation. The strongly FSHr-positive label in the oocytes within Pre-previtellogenic follicles was synchronized with the significant elevation of ovarian cAMP detected at 300 dph, which suggested that diplotene arrest maintenance during early vitellogenesis might be FSH dependent. In addition, preferential selection in asynchronous meiotic initiation has been supposed to act on somatic supportive cells and not directly on germ cells via regulation of FSH that in turn affects downstream estrogen levels. This suggestion was verified by the reciprocal stimulating effect of FSH and E2 on the accelerated meiotic marker SYCP3 and by the inhibited cell apoptosis demonstrated in ovarian cell culture in vitro.ConclusionThe corresponding results contribute an expansion of the understanding of physiological processes and shed some light on the specific factors responsible for gonadotropin function in the early folliculogenesis of crocodilians.